Colloids and Surfaces B: Biointerfaces 135 (2015) 448–457

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Colloids and Surfaces B: Biointerfaces

journal homepage: www.elsevier.com/locate/colsurfb

Chromenone-conjugated magnetic iron oxide nanoparticles. Toward

conveyable DNA binders
Sameena Yousuf a , Israel V.M.V. Enoch a,b,∗ , Paulraj Mosae Selvalumar a ,
Dhanaraj Premnath c
a
Department of Chemistry, Karunya University, Coimbatore – 641114 Tamil Nadu, India
b
Department of Nanosciences and Technology, Karunya University, Coimbatore – 641114, Tamilnadu, India
c
Department of Bioinformatics, Karunya University, Coimbatore – 641114, Tamilnadu, India

a r t i c l e i n f o a b s t r a c t

Article history: Magnetic nanoparticles can transport drug and possibly target cancer. DNA-binding of ligands loaded in
Received 6 January 2015 dextran coated magnetic nanoparticles, could aid their better target-specific binding. In this work, we
Received in revised form 21 March 2015 report the loading of chromenones onto aminoethylamino-modified dextran coated iron oxide nanopar-
Accepted 20 July 2015
ticles, their loading efficiency, and openness for binding to DNA. The magnetic behavior, the size, and
Available online 28 July 2015
the morphology of the nanoparticles are analyzed. The crystallite size of the magnetic nanoparticles is
around 40 nm. The chromenones are present on the surface of the dextran shell, as revealed by their
Keywords:
cyclodextrin-binding characteristics, which is a new approach in comprehending the accessibility of
Dextran
DNA
the surface-bound molecules by macromolecules. The mode of binding of the chromenones to DNA is
Chromenone not altered on surface loading on dextran shell, although the binding strength is generally diminished,
Magnetic nanoparticles compared to the strength of binding of the free chromenones to DNA.
Iron oxide © 2015 Elsevier B.V. All rights reserved.
␤-Cyclodextrin

1. Introduction dependent anti-oxidant properties and selectively protect normal

On account of biocompatibility, stability in tissue environment,
and resistance to enzymatic degradation, dextran is often cho-
sen for surface-immobilization on magnetic nanoparticles, which
are used to transport drugs [1]. However, magnetic nanoparticles
coated with amino end group-containing dextran show a greater
times greater in-vitro cellular uptake than unmodified dextran-
coated nanoparticles [2]. Amination of dextran renders it viabile to
easily get conjugated with chemical groups [3]. Magnetic nanopar-
ticles with a hydrophilic surface can escape reticulo-endothelial
systems and the uptake by macrophages [4,5]. Moreover, the con-
cept of drug-loading on magnetic nanoparticles can be used to
practically overcome the systemic distribution and non-specificity
of anti-cancer drugs. Surface modification prevents the aggrega-
tion of magnetic nanoparticles and limits non-specific adsorption
of biomolecules. Dextran-coating has been reported to exhibit pH-
∗ Corresponding author.
E-mail address: drisraelenoch@gmail.com (I.V.M.V. Enoch).
cells from free radicals, leaving out cancer cells [6].
Besides the above-mentioned points, the rational design of func-
tionalized nanoparticles as tailor-made structures for biomedical
applications requires exploration of the nanoparticle-to-DNA bind-
ing characteristics [7]. A way to obtain nanoparticles that bind to
DNA is provided by the attachment of drugs onto their surface
and facilitate drug–DNA binding. These bound micro aggregates
can be characterized using their fluorescence and light scattering
properties [8]. This kind of micro-aggregation can be detected and
the chemotherapeutic agent-treated-DNA of tumor can be imaged
and treated, if fluorescent pharmaceutically active compounds are
attached to magnetic nanoparticles. It follows that the presence
of DNA-binding molecules on the surface of dextran–coated iron
oxide magnetic nanoparticles is needed in order to be available for
binding to DNA. This further poses a necessity of the experimen-
tal investigation of (i) whether the chromenone is present on the
surface of the dextran shell and remains accessible to DNA, or it is
buried inside the modified dextran shell of the magnetic nanopar-
ticles, and (ii) whether these magnetic nanoparticle–attached
molecules bind to DNA and, if so, with what binding strength
and efficiency? However, systematic solution phase study of the

http://dx.doi.org/10.1016/j.colsurfb.2015.07.049
0927-7765/© 2015 Elsevier B.V. All rights reserved.
 S. Yousuf et al. / Colloids and Surfaces B: Biointerfaces 135 (2015) 448–457
above binding problem has not been reported in detail. In this
article, we present a detailed spectroscopic exploration of the bind-
ing of a series of chromenone-loaded aminoethylamino modified
dextran–coated iron nanoparticles (CHR-loaded IO-DX nPs) to DNA.
Before studying their interaction with DNA, in order to comprehend
the accessibility of the chromenones, we employ ␤-cyclodextrin as
a fit-and-find detector of their openness to binding. ␤-Cyclodextrin
(␤-CD) is a doughnut-shaped cyclic oligosachcharide containing
a hydrophobic cavity capable of accommodating guest molecules
of appropriate size [9]. The biological activities of chromenones
have been well-documented [10] and the bindings of native
chromenones to ␤-CD and duplex DNA have been reported by
our research group [10–16] (Table 1). This article provides the
possible modes and strengths of binding of the chromenones
viz., 2 -Hydroxyflavanone (2 HF), Hesperidin (HP), Naringin (NR),
7-aminoflavone (7AF), Baicalein (BC), Coumarin153 (C153) and
Coumarin314 (C314) loaded, aminoethylamino-modified dextran-
coated iron oxide nanoparticles to ␤-CD and DNA.
2. Materials and methods
2.1. Chemicals
Calf thymus DNA (ctDNA), purchased from Genei (Merck),
India was used without further purification. ctDNA is supplied in
10 mM Tris-HCl (pH 8.0) and 1 mM ethylene diamine tetra acetic
acid (EDTA). The presence of 1 mM EDTA with ctDNA is used to
prevent nucleases from degrading the DNA. ␤-Cyclodextrin and
dextran (MW 20,000) were purchased from Hi-Media, India. The
chromenones were obtained from Sigma, India. Ferrous chloride
tetrahydrate, ferric chloride hexahydrate, epichlorohydrin, sodium
hydroxide, and ethylene diamine (AR, Aldrich) were used without
further purification.
2.2. Preparation of test solutions.
1 mg/ml ctDNA was dissolved in NaCl solution of 50 mmol dm−3
prior to use, to obtain the concentration of 2.27 × 10−4 mol dm−3
which was calculated from the molar extinction coefficient of
6600 dm3 mol−1 cm−1 at 260 nm. The purity of ctDNA sample was
confirmed with the yield of A260 /A280 of approximately in the range
of 1.8–1.9 (where A represents the absorbance). Stock solutions
of all the compounds were prepared in ethanol and diluted with
doubly-distilled water to prepare test solutions. Double-distilled
water was used throughout the experiments. All the experiments
were carried out at an ambient temperature of (25 ± 2) ◦ C.
2.3. Preparation of iron oxide nanoparticles
Ferrous chloride (2.6 g, 0.5 mol dm−3 ) and 3.3 g of ferric chloride
(1 mol dm−3 ) were dissolved in double distilled water and stirred
for 1 h at 50 ◦ C under nitrogen atmosphere. 40 ml of ammonia solu-
tion was added in very small portions (in the range of 0.1 ml) to
the above solution slowly until the solution turned dark brown.
The formation of iron oxide nanoparticles was confirmed using
UV–vis absorption, IR, SEM, EDX, Raman spectroscopy (SI 1 in the
Supplementary data) and XRD.
2.4. Preparation of dextran-coated iron oxide nanoparticles
Dextran (5 g, MW: 20,000) was dissolved in 20 ml double dis-
tilled water and it was sonicated for 30 min. Ferric chloride (0.65 g,
0.2 mol dm−3 ), ferrous chloride (0.51 g, 0.1 mol dm−3 ) were dis-
solved in double-distilled water. The sonicated dextran solution
was added to the ferrous-ferric solution slowly with constant
stirring. Liquid ammonia (40 ml) was added drop-by-drop to the
449
dextran containing ferrous-ferric solutions and stirred at ≈0 ◦ C. The
yellow precipitate turned to green, orange and finally dark brown
at the gradual addition of ammonia. Then, it was refluxed at 80 ◦ C
for 3 h, and then cooled to room temperature, and the supernatant
solution was decanted. To the dark brown precipitate, 150 ml of
ethanol was added to do the aggregation of the colloidal particles.
The precipitate was centrifuged, washed, filtered, and dried to get
the dextran-coated iron oxide nanoparticles.
2.5. Preparation of aminoethylamino modified IO-DX
nanoparticles
Dextran coated iron oxide (100 mg) was dissolved in 100 ml
water and sonicated for 10 min. 0.035 gm of NaBH4 and 35 ml of
2 M NaOH solution were added to the above solution, sonicated for
10 min and then heated to 60 ◦ C with constant stirring. 20 ml of
epichlorohydrin was added to the above solution in drops under
vigorous stirring for half an hour and kept overnight to form a col-
loid. The colloidal solution was centrifuged and the supernatant
solution was decanted to get the precipitate. Phosphate buffer
solution (40 ml) was used to disperse the precipitate. Aminoethy-
lamino modified dextran–coated iron oxide (IO-DX) was prepared
by adding 35 ml of ethylene diamine to the above precipitate and
keeping at 50 ◦ C for 12 h. It was then cooled to room temperature,
centrifuged, filtered, and dried.
2.6. Preparation of CHR-loaded IO-DX nanoparticles
The conjugation of IO-DX and the CHR molecules was carried
out by the addition of the IO-DX nanoparticles (5 mg) to 48, 122,
116, 47, 54, 62 and 63 mg of 2 -Hydroxyflavanone, hesperidin,
naringin, 7-aminoflavone, Baicalein, Coumarin 153 and Coumarin
314 respectively, in ethanol. In a typical procedure, the mixture was
stirred vigorously at 50 ◦ C for 30 min and kept at room temperature.
The solution was centrifuged to get the chromenone conjugated
IO-DX nanoparticles and the UV absorption of the supernatant solu-
tion was measured in order to find the non-loaded chromenone
molecules left behind in the solution. The photographs of the
solutions of CHR-loaded IO-DX nPs are shown in SI2 in the Supple-
mentary data. The chromenone–conjugated IO-DX nanoparticles
were filtered, crushed to get fine powder, stored at room tempera-
ture.
2.7. Instrumentation
Absorption measurements were done using UV–vis spectropho-
tometer (V-630, Jasco, Japan). Fluorescence spectra were recorded
using a spectrofluorimeter (FP750, Jasco, Japan), equipped with a
150 W xenon lamp for excitation. Both the excitation and the emis-
sion band widths were set up at 5 nm. IR spectra were recorded
with KBr pellets on a Perkin–Elmer spectrometer RXI, USA. Ultra-
sonicator PCI 9L 250H, India was used for sonication. The pH was
measured using an Elico LI 120 pH meter, India. The particle size
distribution of IO, IO-DX, and CHR-loaded IO-DX nPs was evalu-
ated using dynamic light scattering measurements with a Malvern
zetasizer nano ZS90, UK. Raman features of nPs were analysed
by Micro Raman spectrometer-800 (LabRAM HR model), France,
with the Argon Laser (514 nm) of 20 mW. The surface topology of
the chromenones, iron oxide nanoparticles, IO-DX nanoparticles
were imaged using JEOL JSM 6360 scanning electron microscope
(Japan). A minimum accelerating voltage of 15 KeV was used in
EDX spectrometry. The diffraction pattern of the samples were
recorded using a Shimadzu XRD 6000 X-ray diffractrometer (Japan)
using a monochromatic X-ray beam from CuK␣ radiation, operat-
ing at the voltage and current of 40 kV and 30 mA respectively. The
magnetization measurements were done using a vibrating sam-
450 S. Yousuf et al. / Colloids and Surfaces B: Biointerfaces 135 (2015) 448–457
Fig. 1. Efficiency of loading of CHR on IO-DX nPs.
ple magnetometer (Lakeshore 7410, US) at room temperature. XPS
spectra were recorded using an X-ray photoelectron spectrometer
(Omicron) with an Mg K␣ monochromatic X-ray source. Molecular
docking was performed using Schrödinger software to optimize the
interaction of the chosen CHR with modified dextran (SI3).
3. Results and discussion
3.1. Loading efficiency
The CHR-loaded IO-DX samples were centrifuged at 70,000 rpm
for 30 min. The free CHR solutions were used as control. They do not
precipitate at this high speed of centrifuging. The loading efficien-
cies (shown in Fig. 1) are calculated, measuring the concentration
of the remaining supernatant liquid by recording their absorption
spectra. Eq. (1) is applied:
[Complex]Total − [Complex]Supernatant
Loading efficiency =
[Complex]Total
where the term complex refers to the CHR-loaded IO-DX nPs. The
IR spectral data and the EDX spectra are given in SI4 and SI5 in the
Supplementary data.
3.2. Size and magnetic properties of CHR-loaded IO-DX nPs
The size distributions of the IO nPs, IO-DX nPs and the CHR-
loaded IO-DX nPs are shown in SI6 in the Supplementary data.
The average particle size of the freshly prepared IO nPs is around
41.44 nm and dextran coating makes it grow in size up to a few
hundreds of nm as shown in Table 2. The magnetizations of the
IO-DX and the CHR-loaded IO-DX nPs were evaluated using Vibrat-
ing Sample Magnetometer (VSM). The room temperature magnetic
hysteresis loops of the compounds are shown in Fig. 2. The IO-DX
nPs were superparamagnetic, showing the absence of hysteresis
loops (Fig. 2a). Lower magnetization is due to the possible pres-
ence of phases that are less magnetic, viz., surface characteristics
and coatings which are attributed to several mechanisms, includ-
ing spin-canting at the particle surface or a surface layer that is
magnetically ineffective.
The values of the magnetization, the coercivity (HC ), the rem-
nant magnetization (MR ), and the saturation magnetization (MS ),
of the free- and CHR loaded- IO-DX nPs are listed out in the Table 2.
The coercivity (Hc ) is strongly influenced by the property of pinning,
which is a main source of coercivity [17]. The domain wall width
is exceeded by the size of the material in the case of bulk samples.
Here, domain wall moves and as a result, magnetization reversal
occurs. The domain walls become pinned at the grain boundaries.
For them to continue moving, an additional energy is needed [18].
In such a case,
√
AK
HC = p1 × d−1 (2)
Js d
HC gets increased due to the creation of more pinning sites, which is
more likely in reduced grain sizes. But in the case of nanoparticles,
a theory different from the above is applicable. It predicts that
K 4 d6
HC = p2 × d6 (3)
Js A
where p2 is another factor. In the case of magnetite,
K = 1.35 × 104 J/m3 A = 10−1 J/m, and the estimated exchange
length is dex = 27 nm. The coercivity HC of the nanoparticle
becomes zero when the particle size comes close to ds (volume
Vs = 25 kT/k) [19], assuming the time of the experiment as 100 s.
The particles are superparamagnetic in an external field. As men-
tioned earlier, the exact size of the synthesized nanoparticles is
41.44 nm, and this size is close to ds . The magnetic behavior of the
particles of free and CHR- loaded IO-DX nPs are reflected in the
room temperature hysteresis loops of the compounds as shown in
the Fig. 2. The uncoated IO nanoparticles are superparamagnetic
and the sample’s magnetization curve shows the absence of any
hysteresis loop. This magnetic behavior is slightly different in all
the CHR-loaded IO-DX nPs. Thin hysteresis loops are observed.
In general, a change of magnetization occurs due to the surface
characteristics and coatings, which can be attributed to a variety
of mechanisms [20]. A surface layer that modifies the magnetic
behaviour of nanoparticles is not uncommon. However, the mag-
netic behaviour of a nanomaterial is difficult to be predicted by a
simple approach, if the substance contains more than one type of
atom. Indeed, the magnetic moments of electrons which constitute
the electron structure of atoms and molecules, and hence the crys-
tals, are the fundamental sources of material magnetism. But, the
particle’s shape, the morphology, the surface, and the composite
× 100(1)
nature, etc [21] do play roles in the net magnetic behavior of the
nanoparticles. The outer boundary of a particle is translation-
ally less periodic, the co-ordination number is reduced, and the
magnetic exchange bonds of the surface atoms are broken, in the
case of coated magnetic nanoparticles. These influence the surface
effects [22]. The physical properties of the surface and the core of
nanoparticles can be different. Hence, the magnetic parameters of
a coated nanoparticle as a whole are determined by the magnetic
co-operation or competition between the surface and the core
magnetic sub-system. It is an over-simplification to present a
magnetic nanoparticle as a single super-large magnetic moment
[22]. In our experiment, we observe that the superparamagnetic
behaviour of the IO-DX nPs is not affected on dextran coating.
It remains superparamagnetic, similar to the uncoated, freshly
prepared IO nPs. The differences in the saturation magnetizations
(MS ) of the CHR-loaded IO-DX correspond to the pronounced
surface effect in these samples and the role of their morphology.
Moreover, the modified dextran shell also might slightly unwind
on CHR-loading, which could lead to the changes in the extent of
exposure of the IO nPs to the solvent dipoles. However, we did not
observe peptization of the nanoparticles which, if occurred, would
manifest in the absorption spectra. Also, there was no turbidity
or precipitation from the test solutions, implying that the IO-DX
nPs are well-dispersed in solution and the photographs of these
solutions are shown in SI7.
The X-Ray Diffraction patterns of the magnetic IO-DX and free
CHRs and CHR loaded- IO-DX compounds are shown in Fig. 3. The
procedure of crystallite size calculation is given in SI8. The crystal-
lite sizes and the strain in the crystals are given in Table SI8 in the
Supplementary data. The results are consistent with the observa-
tion that the IO nPs display crystalline Fe3 O4 structure. The peaks
 S. Yousuf et al. / Colloids and Surfaces B: Biointerfaces 135 (2015) 448–457 451

Fig. 2. Vibrating sample magneto metric spectra of free- and CHR-loaded IO-DX nPs (a) free IO-DX; and IO-DX conjugated with (b) 2’-Hydroxyflavanone, (c) Hesperidin, (d)
Naringin, (e) 7-Aminoflavone, (f) Baicalein, (g) Coumarin 153, (h) Coumarin 314.

Fig. 3. X-ray diffraction pattern of (a) free- and IO-DX nPs loaded with (b) 2’-Hydroxyflavanone, (c) Hesperidin, (d) Naringin, (e) 7-Aminoflavone, (f) Baicalein, (g) Coumarin
153, (h) Coumarin 314.
452 S. Yousuf et al. / Colloids and Surfaces B: Biointerfaces 135 (2015) 448–457
are in agreement with the standard card of magnetite PDF No. 01-
071-6337. The noise observed at the background of the figures is
due to the amorphous dextran polymer shell. The crystallite sizes
of the modified IO nPs fall in the range of 20 to 43 nm. Intense
reflections are present at 2␪ values ≈25◦ corresponding to the
experimental d–spacing for iron of ≈ 3.7 Å and hence, it is observed
a high percentage of a single phase of Fe3 O4 is present. The crys-
tallite size is found very close to the physical size of the IO nPs,
which suggests that polycrystalline nanoparticles are not formed.
The Scanning Electron Microscopic images of the freshly prepared
IO, IO-D, IO-DX and various CHR-loaded IO-DX nPs are compared
with those of the free CHRs and shown in Fig. 4. The colloidal IO
nPs show randomly oriented irregular shaped structures due to the
anisotropic dipole–dipole interactions [23]. Dextran coating and
attachment of compounds increase the net size of the IO-DX struc-
tures and, among most of them, quasi-linear cuboid or rod–like
structures are observed. Lateral attraction between long chains of
folded dextran sub-structures induces a self-organization and an
entropy–minimized, favored pattern formation on the surface of IO
nPs. The striking differences in the morphology between the free-
and the CHR-loaded IO-DX samples is an indication that the surface
of the IO-DX is altered at the addition of various compounds. The
average diameters of the ordered patterns of CHR-loaded IO-DX
particles are in the range of a few hundreds of nanometers.
3.3. Binding of CHR-loaded IO-DX nPs to ˇ-CD
In general, between the free and the IO-DX-attached com-
pounds, there is no appreciable shift of absorption band at the
addition of ␤-CD (The significant absorption spectral data of the
CHR-loaded IO-DX nanoparticles, detailing the wavelengths of
spectral bands and the shift of wavelengths on the addition of ␤-CD
are given in SI9). This trend is quite commonly observed in bands
corresponding to ␲→␲* (shorter wavelength band) and n→␲* tran-
sitions (longer wavelength bands). The shifts of absorption bands of
the IO-DX-attached compounds from those of the free unmodified
compounds, on ␤-CD complex formation, are similar. However, in
the case of Coumarin 314, we observed a remarkable difference.
The 448 nm band of the free Coumarin 314 shifts to 454 nm on ␤-
CD complexation, whereas the 444 nm band of the IO-DX-attached
Coumarin 314 shifts to 409 nm when ␤-CD is added. These oppos-
ing red vs. blue shifts suggest that different kinds of interactions
exist between ␤-CD and the free- and IO-DX-attached Coumarin
314.
Using the absorption spectral data of the guest molecules (form-
ing the host–guest complexes), the plots of 1/AS –A0 vs. 1/[␤-CD] of
various complexes are made, following the Eq. (4) [24]:
1 1 1 1
=  +   
As − A0 A − A0 A − A0 K ˇ − CD
in order to determine the stoichiometry and the binding constants
of the complexes.
In the above equation, A0 refers to the absorbance of each one
of the chromophores (IO-DX-attached compounds) in water, AS
is the absorbance at different concentrations of the added ␤-CD,
and A’ is the absorbance at the highest concentration of ␤-CD. Lin-
ear plot of 1/ AS –A0 vs. 1/[␤-CD], in the case of any of the guest
molecules, leads to the inference that a 1:1 host–guest complex is
formed. The binding plots made using absorption spectral data are
shown in the insets of Fig. 5. Indeed, we observed a linearity per-
taining to the formation of 1:1 complexes in all the IO-DX-attached
compounds. A notable result is obtained in a structurally similar
molecule, Naringin. The native form of Naringin forms a 1:2 ␤-
CD complex, i.e., two ␤-CD molecules bind one Naringin molecule.
However, the IO-DX-attached Naringin binds to only one ␤-CD
molecule because the bulky dextran blocks one of the binding sites
of Naringin, i.e., the chromen-4-one moiety, as its carbonyl group
is less available on attachment to dextran for binding to ␤-CD. Only
the phenolic moiety is available for binding to ␤-CD and hence the
1:1 complex is formed.
The binding of the IO-DX-attached compounds to ␤-CD is
studied also using fluorescence titration (shown in SI10), as flu-
orescence spectroscopy is a highly sensitive technique. The scope
of the fluorescence titration is to derive information on the stoi-
chiometry and the binding constant of the host–guest complexes.
The binding plots are also shown in insets of figures in SI10.
In all the cases except Coumarin 314, we observe fluorescence
enhancement when ␤-CD is added. For a simple 1:1 host–guest
complex, the I/I0 varies with the added ␤-CD according to the fol-
lowing Eq. (5) [25]:
I Imax K[CD]0
=1+ (5)
I0 (I0 − 1) 1 + K[CD]0
where Imax /I0 is the maximum enhancement of fluorescence and K
is the binding constant. A double-reciprocal plot of 1/Imax − I0 vs.
1/[␤-CD] shows linearity for a 1:1 complex. In the case of 1:2 com-
plex, the concentration of ␤-CD is squared and 1/[␤-CD]2 is taken
along the X axis and the plot is linear if the stoichiometry is 1:2. In
the fluorescence titration of the CHR-loaded IO-DXs against ␤-CD,
there are wavelength shifts of fluorescence maxima and enhance-
ment of fluorescence. The accommodation of guest molecules in
the ␤-CD cavity exerts a restriction on the intra-molecular rota-
tional and vibrational freedom of the guest molecule. Also, the local
polarity experienced by the guest molecule inside the hydrophobic
cavity is much smaller than that in the aqueous solution. This leads
to a significant destabilization of the relative polar S1 ground state
and a smaller destabilization of the less polar S0 ground state. This
effect results further in a significantly larger S1 − S0 energy gap of
the encapsulated guest molecule [26]. Hence, the fluorescence of
the guest molecule is blue-shifted. The fluorescence enhancement
is a consequence of the increased energy gap, which reduces the
probability of the non-radiative decay and a corresponding increase
in the fluorescence quantum yield [27]. For reasons given in the
preceding paragraphs on the absorption spectra of Naringin, the
fluorescence titration of this compound against ␤-CD shows a 1:1
stoichiometry. The stoichiometry and binding constants of all the
host–guest complexes of the CHR-loaded IO-DXs are listed out in
Table 1.
The stoichiometry is unaltered in all the cases except in
Naringin. From the reported binding constants of the free CHR-
␤-CD complexes, the binding constants of the CHR-loaded IO-DX
nPs show a marked decrease in their magnitudes. The binding con-
stant is dependent on the fit of the guest molecule inside the ␤-CD
cavity as well as its polarity, and any other type of bonded inter-
(4)
action in the complex. Since the polarity experienced by the native
compounds is different from that of their IO-DX-attached forms,
there occurs a different rate of enhancement of fluorescence. From
the observations in the absorption and fluorescence titrations, the
striking point seen is that when the compounds are loaded onto
IO-DX, they still bind to ␤-CD and hence are locally accessible from
outside the IO-DX surface. However, the binding constant values
are lowered, which implies that the strength of binding is altered.
This again is a reflection on the accessibility of IO-DX-attached
compounds to ␤-CD. Spectroscopic evidences suggest that the com-
pounds, when attached to IO-DX, are loosely bound on the surface
of the dextran shell covering the magnetic nanoparticle.
3.4. Interaction of CHR-loaded IO-DX nPs with DNA
The CHR-loaded IO-DX nPs display hyperchromic shifts of
absorption bands with little blue shifts (Fig. 6). Single isosbestic
 S. Yousuf et al. / Colloids and Surfaces B: Biointerfaces 135 (2015) 448–457 453

Fig. 4. SEM images of (a) naked Iron oxide, (b) Iron oxide coated with dextran, (c) aminoethylamino modified dextran-coated iron oxide nanoparticles, (d) dextran, and free
and IO-DX nPs attached chromenones viz., (e–f) 2 -Hydroxyflavanone, (g–h) Hesperidin, (i–j) Naringin, (k–l) 7-Aminoflavone, (m–n) Baicalein, (o–p) Coumarin 153, (q–r)
Coumarin 314.

points are absent in some of the compounds, indicative of the
absence just one mode of binding. In general, most of the com-
pounds display absorption characteristics similar to unmodified
native compounds, on DNA binding. The shifts of absorption bands
are listed out in SI11. There is a general decrease in the binding
strength of the CHR-loaded IO-DXs, from the binding strengths
of native chromenones. However, larger binding constant values
of IO-DX-attached Naringin, and Coumarin 153 are observed on
DNA binding, compared to their corresponding native forms. An
increase in the binding constant value is an indication that the IO-
DX attachment does not hinder their binding to DNA. The notable
general observations are (i) the IO-DX attachment of the com-
pounds does stop their binding to DNA and (ii) dextran does not trap
the IO-DX-attached compounds into their possibly coiled strands;
instead, these are available on the surface of the IO-DX and are
accessible to DNA. The binding of CHR-loaded IO-DXs to DNA is
also studied using fluorescence spectroscopy (SI12). Coumarin 153
and Coumarin 314 show quenching of fluorescence on the addition
of aliquots of DNA, keeping the concentration of the fluorophores
fixed. The quenching of fluorescence is an indication of the bind-
ing interaction between each of these compounds and DNA. The
Stern–Volmer quenching constants (KSV ) are calculated from the
fluorescence titration data using the Eq. (6) [28]:
F0
= 1 + KSV [Q ] = kq 0 [Q ] (6)
F
where F0 and F are the intensities of fluorescence of the flu-
orophores in the absence and the presence of DNA, kq is the
bimolecular quenching constant, and ␶0 is the fluorescence lifetime.
The KSV values are listed out in Table 1.
The fluorescence spectra of the IO-DX-attached Naringin and 7-
aminoflavone show insignificant changes at the addition of DNA,
suggesting that their binding to DNA is weak and it occurs in the
ground state, as shown by the absorption spectra. Baicalein, and
Coumarin 314 show enhancement of fluorescence at the addition
of DNA. These show that the binding of these IO-DX-attached com-
pounds to DNA takes place. This is thought to occur due to the
454 S. Yousuf et al. / Colloids and Surfaces B: Biointerfaces 135 (2015) 448–457

Fig. 5. Absorption spectral titrations of the IO-DX-compounds–␤-CD binding (a) 2 -Hydroxyflavanone, (b) Hesperidin, (c) Naringin, (d) 7-Aminoflavone, (e) Baicalein, (f)
Coumarin 153, (g), Coumarin 314.
 S. Yousuf et al. / Colloids and Surfaces B: Biointerfaces 135 (2015) 448–457 455

Fig. 6. Absorption spectral titrations of the CHR–loaded IO-DX–DNA binding (a) 2 -Hydroxyflavanone, (b) Hesperidin, (c) Naringin, (d) 7-Aminoflavone, (e) Baicalein, (f)
Coumarin 153, (g) Coumarin 314.
456 S. Yousuf et al. / Colloids and Surfaces B: Biointerfaces 135 (2015) 448–457

Table 1
Binding parameters of CHR-loaded IO-DX nPs– and free CHRs-binding to ␤-CD and DNA.

Stoichiometry and binding constants of CHR-loaded IO-DX nPs–␤-CD association

Compound CHR- loaded IO-DXs Free CHRs*

UV Fluorescence UV Fluorescence

St. Binding Constant St. BindingConstant St. Binding Constant St. Binding Constant

2’HF 1:1 23 M−1 1:1 47 M−1 1:1 40.54 M−1 1:1 489 M−1
HP 1:2 6.66 × 104 M−2 1:2 1.46 ×104 M−2 1:2 2.84 × 104 M−2 1:2 9.63 × 103 M−2
NR 1:1 94 M−1 1:1 27 M−1 1:2 3.97 × 106 M−2 1:2 8.68 × 104 M−2
7AF 1:1 278 M−1 1:1 290 M−1 1:1 455 M−1 1:1 150.41 M−1
BC 1:1 69 M−1 1:1 36 M−1 1:1 117 M−1 1:1 291.88 M−1
C153 1:2 9.71 × 103 M−2 1:2 1.49 × 104 M−2 1:2 57.74 × 103 M−2 1:2 95.53 × 103 M−2
C314 1:1 84.49 M−1 1:1 202 M−1 1:1 1895.12 M−1 1:1 Ka =257 M−1

K and KSV values of free CHRs and CHR-loaded IO-DXs on DNA binding

Compound CHR- loaded IO-DXs Free CHRs*

UV Fluorescence UV Fluorescence
K (M-1 ) KSV (M-1 ) K (M-1 ) KSV (M-1 )

2’HF 1.09 × 104 – 1.87 × 105 1.87 × 105

HP 5.42 × 104 – 4.47 × 104 1.01 × 105
NR 1.69 × 105 – 8.22 × 104 4.70 × 104
7AF 5.73 × 103 – 1.06 × 105 –
BC 6.37 × 104 – 2.22 × 103 9.55 × 104
C153 5.70 × 104 1.51 × 104 3.96 × 104 1.98 × 105
C314 6.25 × 104 4.42 × 104 1.03 × 105 –
*
References [11–16] and [31–33]. Note: St.: Stoichiometry.

Table 2
Particle size, magnetic properties, and crystallite size of free- and CHR-loaded IO-DX nPs.

Sample Particle diameter d (nm) Coercivity Hc (G) MS (emu/g) MR (emu/g) Crystallite size in nm with Strain ()

IO-DX nPs 723.7 0.65 0 0 45 (0.0003)

CHR- loadedIO-DX nPs 2 HF 540.2 321.84 4.06 1.25 27 (0.0004)
HP 1279.0 274.96 2.05 0 24 (0.0013)
NR 960.1 275.79 1.27 0.24 19 (0.0016)
7AF 1255 290.24 0.59 0.21 19 (0.0016)
BC 376.0 275.91 42.33 10.92 23 (0.0010)
C153 860.5 312.91 29.49 9.11 39 (0.0005)
C314 662.3 313.52 34.77 10.95 34 (0.0007)

Scheme 1. Summary of the experimental procedures involved and the various events of binding.

change of micro-environmental polarity through the removal of
water molecules by intercalation [29,30].
The binding of ligands to DNA can be tuned by ␤-
CD–encapsulation of the compounds [31–33]. When ␤-CD partially
covers up a guest molecule, the part of the molecule outside the ␤-
CD cavity binds to DNA. We applied this method to the CHR-loaded
IO-DXs in their ␤-CD–bound form. When in ␤-CD–covers up the
compounds attached to IO-DX, they cannot interact with DNA and
their fluorescence does not change on the addition of DNA (a few
representative cases are shown in SI13). This is an evidence for the
availability of the compounds on the surface of IO-DX, which ren-
ders them accessible by DNA for binding, and inaccessible when
encapsulated by ␤-CD. The experiments and inferences discussed
above are represented in the Scheme 1.
 S. Yousuf et al. / Colloids and Surfaces B: Biointerfaces 135 (2015) 448–457
4. Conclusions
We report the loading of chromenones onto aminoethy-
lamino modified dextran-coated superparamagnetic iron oxide
nanoparticles and their binding to ␤-cyclodextrin and ctDNA.
The chromenones get loaded onto the shell of modified dextran,
with the involvement of hydrogen bonding between the modi-
fied dextran and the chromenones. This conjugation, weaker than
covalent bonding, is best for delivery of drugs. They are acces-
sible by ␤-cyclodextrin in the formation of inclusion complexes.
The strengths of ␤-CD complexes of CHRs surface-bound to IO-
DX nPs are lesser when compared to the free chromenones’ ␤-CD
complexes. The chromenone-loaded IO-DX nanoparticles are kept
intact in solution and do not undergo precipitation within the
studied concentration range. The surface–loaded chromenones are
able to bind to DNA, although with a lesser binding strength than
the free chromenone–ctDNA binding. Anticancer drug delivery
using iron oxide hinges on the possible directed transport of the
magnetic nanoparticles into tumor cells. The drug delivery using
drug–nanoparticle binding plays a vital mode of drug targeting,
it can be utilized applying the idea of loading of DNA-interacting
molecules onto magnetic nanoparticles and delivering them into
cancer cells, by directing using an external magnetic field. Our work
has explored such an important area in order to comprehend the
behavior of drug-loaded IO-DX nanoparticles as binding agents.
Acknowledgments
We thank our Vice-Chancellor Dr. S. Sundar Manoharan, for
his efforts in establishing our new research lab. The funding by
the DST, Government of India, is acknowledged (Project: SR/FT/CS-
062/2009).
Appendix A. Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.colsurfb.2015.07.
049
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