Antioxidant flavone analog functionalized fluorescent silica nanoparticles: Synthesis and exploration of their possible use as biomolecule sensor

Article abstract of DOI:10.1016/j.colsurfb.2017.05.074

For the first time, a synthetic fluorescent antioxidant flavone analog was successfully anchored onto the surface of the APTES-modified mesoporous silica nanoparticles (NPs) through sulfonamide linkage. The surface chemistry and morphology of the flavone

Full text of DOI:10.1016/j.colsurfb.2017.05.074

Accepted Manuscript
Title: Antioxidant flavone analog functionalized fluorescent
silica nanoparticles: Synthesis and exploration of their
possible use as biomolecule sensor
Authors: Sreeparna Das, Shaikh Batuta, Md. Niharul Alam,
Chandrani Fouzder, Rakesh Kundu, Debabrata Mandal,
Naznin Ara Begum
PII:
S0927-7765(17)30342-9
DOI:
Reference:
http://dx.doi.org/doi:10.1016/j.colsurfb.2017.05.074
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Colloids and Surfaces B: Biointerfaces
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13-2-2017
23-5-2017
29-5-2017
Please cite this article as: Sreeparna Das, Shaikh Batuta, Md.Niharul Alam,
Chandrani Fouzder, Rakesh Kundu, Debabrata Mandal, Naznin Ara Begum,
Antioxidant flavone analog functionalized fluorescent silica nanoparticles: Synthesis
and exploration of their possible use as biomolecule sensor, Colloids and Surfaces B:
Biointerfaceshttp://dx.doi.org/10.1016/j.colsurfb.2017.05.074
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Antioxidant flavone analog functionalized fluorescent silica nanoparticles:
Synthesis and exploration of their possible use as biomolecule sensor
Sreeparna Dasa, Shaikh Batutaa, Md. Niharul Alama, Chandrani Fouzderb, Rakesh Kundub,
Debabrata Mandalc and Naznin Ara Beguma*
aDepartment of Chemistry, Visva-Bharati (Central University), Santiniketan 731 235, India.
bDepartment of Zoology, Visva-Bharati (Central University), Santiniketan 731 235, India.
cDepartment of Chemistry, University College of Science and Technology, University of
Calcutta, 92, Acharya Prafulla Chandra Road, Kolkata 700 009, India.
 Corresponding AuthorE-mail address: naznin.begum@visva-bharati.ac.in (N. A. Begum) Tel.:
+91-9434431810; Fax: +91 3463261526.
Graphical abstract
1

Highlights
 Antioxidant flavone modified fluorescent silica nanoparticles (NPs) synthesis.
 Anchoring of a fluorescent synthetic antioxidant flavone analog on NP surface.
 Mesoporous silica NP matrix is tagged with flavone through sulfonamide linkage.
 These NPs are biocompatible, retain their intrinsic fluorescence in cellular medium.
 These fluorescent NPs can have the potential to probe biomolecules, like BSA.
ABSTRACT
For the first time, a synthetic fluorescent antioxidant flavone analog was successfully anchored
onto the surface of the APTES-modified mesoporous silica nanoparticles (NPs) through
sulfonamide linkage. The surface chemistry and morphology of the flavone modified fluorescent
silica (FMFS) NPs were studied in detail. The flavone moiety when attached onto the FMFS NP
surface, imparted its characteristic fluorescence and antioxidant activities to these NPs.
Moreover, the NPs are highly biocompatible as evidenced from their cytotoxicity assay on
normal lung cell (L132). The fluorescence activity of these biocompatible NPs was further
utilized to study their interaction with a biomolecule, BSA (Bovine Serum Albumin). It was
interesting to note that the fluorescence behavior of FMFS NPs completely changed on their
binding with BSA. On the other hand, the intrinsic fluorescence activity of BSA was also
significantly modified due to its interaction with FMFS NPs. Thus, the sensing and detection of
biomolecules like BSA in presence of FMFS NPs can be accomplished by monitoring changes
in the fluorescence behavior of either FMFS NPs or BSA. Furthermore, these FMFS NPs
retained their intrinsic fluorescence behavior in the cellular medium which opens up their
possible use as biocompatible cell imaging agents in future.
Keywords: Antioxidant flavone analog, Fluorescent silica nanoparticles, Biocompatible,
Biomolecule sensor, BSA.
2

1. Introduction
Owing to their unique structure dependent optical and electronic properties, nanoparticles (NPs)
have found several technological applications spanning topics as diverse as photonics, catalysis,
biological fluorescence sensing/imaging, etc [1-5]. In this work, we deal specifically with silica
NPs, which are characterized by characteristic structural features like high surface to volume
ratio, large and uniform pore dimensions, and easily controllable particle size [1-5]. Although
silica NPs have no intrinsic fluorescence, it is possible to incorporate extrinsic fluorophores into
them, generating fluorophore modified fluorescent silica NPs. One of the cherished goals of
silica nanochemistry is the synthesis of such modified fluorescent silica NPs designed to carry
out highly specific tasks. Incidentally, fluorophore modification of silica NPs to a given degree
of precision is much easier as compared to that of other classes of NPs like metal NPs and
quantum dots.
In biological and medical fields, various types of fluorescent nanomaterials are used as drug
delivery vehicles [6-7], fluorescent biomarkers for diseased cells and as image contrast agents for
cancer diagnostics [8-11]. In such applications, the performance of appropriately fluorophore
modified fluorescent silica NPs often outweighs those of the metal NPs and quantum dots in
terms of various critical factors like hydrophilicity, sensitivity, photo stability, biocompatibility
and tolerance in living systems [12,13].
Incorporation of the extrinsic fluorophore can be accomplished in two ways: either by
encapsulating it inside the silica matrix, or by attaching it onto the surface of the silica NPs
through appropriate functionalization process [1, 3-4, 14-16]. Although many different
fluorophores have been incorporated in this manner, most of them are synthetic, and have no
worthwhile role in the natural/ biological world [14-16]. Hence, the resultant fluorescent silica
NPs are unlikely to be biocompatible.
Keeping this in mind, we have attempted the synthesis of flavone modified fluorescent silica
(FMFS) NPs, where the extrinsic fluorophore has been chosen to be a flavone type of
compound. Flavones and flavonols belong to the flavonoid class of naturally occurring dietary
antioxidants (Fig.1), found extensively in plants. These compounds are known to fight against
reactive oxygen species (ROS) induced oxidative stress related neuro-degenerative and
cardiovascular diseases [17-22]. Besides that, these classes of naturally occurring compounds
show a wide spectrum of biological and pharmaceutical activities as anti-inflammatory, chemo-
3

preventive and anticancer agents [22-23]. Flavones (having a vast range of structures and
activities) can be isolated from their natural sources (plants) and interestingly they can also be
synthesized easily in the laboratory using very simple and easy laboratory set-up [24-26].
Many naturally occurring flavones and their synthetic analogs are fluorescent [17-19]. Very
often, these types of compounds are photo-stable and have high quantum yield [27]. Hence, these
compounds are suitable as fluorophores and thus they can be explored for in-vivo cell imaging or
they can be used as disease biomarkers. In both the cases, it is necessary that they must retain
their intrinsic fluorescence behavior in the cellular medium. But flavones in their free form are
poorly soluble in water which reduces their bioavailability in aqueous medium like interior of a
cell [28]. Attempts have been made to incorporate various suitable functional groups in the
parent moiety of the flavones in order to enhance their aqueous solubility [29]. Incidentally, such
chemical modifications of flavone structures can also be explored to tag these fluorescent
molecules on appropriately modified silica NP surface, leading to the fabrication of
biocompatible fluorophore modified fluorescent silica NPs. These NPs may have the potential to
be used for in-vivo cell imaging and for the development of other biomedical diagnostic and
therapeutic tools. Moreover, these hybrid NPs have the combined advantages associated with the
best features of each of the components involved, such as (i) the antioxidant effect of flavone
moiety and (ii) tailor-made structural and hydrophilic properties of silica NPs, essential for
targeted cellular activity.
In our previous work, it was found that 3-hydroxyflavones having an electron rich substituent at
the 4'-position of ring-B (Fig.1) showed high antioxidant activity [23] as well as strong
fluorescence intensity in the wavelength region of 490-517 nm [27, 30,17-18]. Hence, for the
present work 3-hydroxy-4'-methoxyflavone (2) (Fig.1) was synthesized and (2) was derivatized
suitably so that it can be grafted on the surface of the 3-aminopropyl-triethoxysilane (APTES)
modified mesoporous silica NPs through sulfonamide bond. These flavone modified fluorescent
silica (FMFS) NPs showed high antioxidant activity (in-vitro) in aqueous medium. Moreover,
these NPs retained their intrinsic and intense fluorescence activity in cellular environment
[human lung cancer cells (A549)].
In this work, the interactions of FMFS NPs and BSA [bovine serum albumin, a globular carrier
protein having approximately 76% sequence homology with human serum albumin (HSA)] was
spectrophotometrically monitored by tracking the change of fluorescence behavior of FMFS
4

NPs in presence of BSA or vice-versa. Biocompatibility of FMFS NPs was tested by in-vitro
erythrocyte induced hemolysis and MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
bromide] assays.
Figure 1
2. Experimental section
For details of the chemicals and instruments used in the present work please see the Supporting
Information, page S3-S5.
2.1. Method of synthesis of fluorescent flavone analog, 3-hydroxy-4-methoxyflavone (2)
and its derivatization (sulphonation) to 3,4'-dimethoxy-3'-flavone sulfonic acid (3)
2'-Hydroxy-4-methoxychalcone (1) was used as the precursor of (2). (1) was synthesized by
Claisen-Schimdt reaction [23-26] between appropriately substituted acetophenone (a) and
benzaldehyde (b) derivatives (Scheme1). The chalcone (1) was converted to corresponding
flavone (2) by Algar-Flynn-Oyamada reaction [23, 26] (Scheme1).
Flavone (2) was grafted on the surface of APTES-modified silica NPs through sulfonamide
linkage. For this, (2) was sulfonated according to the method of Silva et al. [31] with
modifications (Scheme1). The presence of hydroxyl group at 3-position of the flavone moiety
created obstacle during its covalent linking on the silica surface. To avoid this, the sulfonated
flavone was methylated at its 3-OH position to get 3,4'-dimethoxy-3'-flavone sulfonic acid (3).
This was covalently linked on the silica surface by sulfonamide bond.
Scheme 1
Detailed Method:
268 mg of (2) (1 mmol) was added to ice-cold chlorosulfonic acid (0.41 ml; 6.11 mmol) in small
parts and the reaction mixture was stirred continuously. After the completion of HCl evolution,
the reaction mixture was taken out from ice-bath and allowed to stand for a while so that it could
attain room temperature. This mixture was stored in an air-tight container. This container was
kept in moister free place under N2 atmosphere to prevent aerial oxidation of the reaction
mixture. The reaction mixture was kept in this condition for three days. Then, it was slowly
poured into crushed ice with continuous stirring whereby, a yellow precipitate was obtained.
This precipitate was collected and washed with ice-cold water several times to eliminate the
excess acid. However, no attempt was made to make it fully acid free. The precipitate having pH
5

3 was collected and dissolved in methanol. This methanol solution was refluxed for 2 hours.
Upon cooling, this solution gave brown colored crystals of 3,4-dimethoxy-3'-flavone sulfonic
acid (3). The crystals were separated from the reaction mixture followed by its characterization.
In each step, the purity and yield of the products were routinely checked. Structures of all the
synthesized products were confirmed on the basis of m.p. and spectroscopic data (Supporting
information, page S5-S7).
2.2. Synthesis of flavone modified fluorescent silica (FMFS) NPs
Flavone analog (3) was used to synthesize FMFS NPs. The silica NPs was functionalized with 3-
aminopropyl-triethoxysilane (APTES) according to a previously reported method [32-33] prior
to the grafting of (3) on its surface.
Scheme 2
APTES conjugated silica NPs were synthesized by adding 10 mL APTES to a suspension of
pretreated dry silica (Aerosil 200, 10g) in 50 mL toluene. This suspension was refluxed at 80oC
for 24 hours and the solvent was evaporated under reduced pressure to obtain the amine
functionalized silica NPs (APTES-silica), this was further purified by repeated rinsing with a
series of solvents (toluene, ethanol and acetone) and dried in vacuum at 80oC for 12 hours [34-
35]. APTES-silica NPs have aminopropyl functional group on their surfaces which was exploited
to synthesize FMFS NPs. Flavone analog (3) was conjugated with APTES-silica system by the
formation of sulfonamide bonds between the surface active amine groups of the later and the
sulfonic acid groups of (3) (Scheme 2).
The method is discussed here in brief: 0.1 mL triethylamine was added to a suspension of 1g of
amine functionalized silica (APTES-silica) in 30 mL toluene. After that, a 1.8 mmol of (3) was
added to it and mixed well. The reaction mixture was refluxed for 12 hours at 80oC whereby a
solid product was obtained. This solid mass was washed with a series of solvents (toluene,
ethanol and acetone). After that, it was dried in vacuum at 80oC for 12 hours to get the final
product i.e. flavone modified fluorescent silica (FMFS) NPs. The surface chemistry and
morphology of these FMFS NPs were further confirmed on the basis of Fourier transform
infrared (FT-IR) spectroscopy, thermo gravimetric analysis (TGA), N2 adsorption isotherm,
dynamic light scattering (DLS), zeta potential and transmission electron microscopy (TEM)
studies.
2.3. Biocompatibility and bioavailability assays of the FMFS NPs in aqueous medium
6

One of the major targets of this study was to evaluate the biocompatibility of silica NP after its
functionalization with fluorescent synthetic flavone analog and at the same time, to study the
beneficial effect of FMFS NPs, if any. The bioavailability of the hydrophobic antioxidant
synthetic flavone analog in aqueous medium was checked after its attachment on the silica NP
surface. The beneficial effect of FMFS NPs and bioavailability of flavone moiety attached on its
surface were assessed in terms of in-vitro antioxidant activity of these NPs in aqueous medium
which was measured by ferric reducing antioxidant power (FRAP) assay. In-vitro erythrocyte
induced hemolysis and MTT assays of FMFS NPs were performed to check their bio-
compatibility.
2.3.1. Method of ferric reducing antioxidant potential assessment of FMFS NPs
In the present case, the in-vitro antioxidant activity of FMFS NPs and the synthetic flavone
analog (2) was studied by ferric reducing antioxidant potential assay method of Oyaizu (1986)
with little modification [36]. Gallic acid was used as standard antioxidant. Detailed method is
discussed in Supporting Information, page S8.
2.3.2. In-vitro erythrocyte induced hemolysis assay of FMFS NPs
Blood compatibility is considered to be a simple and reliable measure for estimating the non-
toxicity and bio-compatibility of chemical compounds. Silica NPs (before and after
functionalization with fluorescent synthetic flavone analog) were tested for their blood
compatibility by in-vitro erythrocyte induced hemolysis assay. Hemolysis experiments were
carried out according to the method of Ravikumara et al. [37] with slight modifications
(Supporting Information, page S10).
2.3.3. MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay and
fluorescence cell imaging in presence of FMFS NPs
MTT assays of 2 and FMFS NPs were done to determine their cytotoxicity in human normal
lung cell (L132) (Supporting Information, page S11).
A fluorescent probe for biomolecules must retain its fluorescence activity in the cellular
environment. So it is very much essential to study whether the intrinsic fluorescence activity of
FMFS NPs can also be observed in the cellular medium or not. Hence, the fluorescence activity
of FMFS NPs along with the precursor fluorescent flavone (2) in cellular medium [human lung
cancer cells (A549)] was monitored with the help of fluorescence microscope. Fluorescence cell
7

imaging with the help of FMFS NPs was done according to the method discussed in supporting
information, page S12.
2.4. Spectrophotometric studies on the interaction of FMFS NPs with bovine serum
albumin (BSA)
20 μM stock solution of FMFS NPs was added to 2.5 mL of Tris-HCl buffer of pH~7.4 taken in
a 3.5 mL cuvette. The absorbance of FMFS NPs was recorded in the absence and presence of
BSA having concentration range of 2-10 μM. UV-Vis spectra were recorded at room temperature
in the wavelength region of 300-450 nm on a dual beam UV-Vis spectrophotometer.
Similarly, the steady-state fluorescence emission spectra of the FMFS NP solution (20 µM) at
λex 280 nm (with a slit width of 5/3 nm) was recorded for the wavelength range of 300-540 nm in
the absence and presence of BSA solutions (0.3-4.8 μΜ) at pH 7.4.
On the other hand, to monitor the change of fluorescence behavior of BSA in the presence of
FMFS NPs, 2.5 mL aliquot of 2 μM BSA in 10 mM Tris-HCl buffer of pH~7.4 was
spectrofluorometrically titrated with FMFS NP solution (2-32 μM). The emission spectra of
BSA solutions were recorded in the wavelength range of 300-540 nm at λex of 280 nm. In case of
recording excitation spectra of BSA solutions, the excitation wavelength range selected was 200-
320 nm and the emission wavelength (λem) was fixed at 347 nm. In both the cases, slit width was
set at 5/3 nm.
The fluorescence quenching and binding parameters regarding the interactions of FMFS NPs
with BSA have been calculated using the appropriate standard equations [38].
The anisotropy (r) of FMFS NPs (20 µM) in presence of BSA solutions of different
concentrations (6-22.8 µM) was determined by the steady-state anisotropic measurements on the
PerkinElmer LS55 spectrofluorometer. The excitation (λex) and emission (λem) wavelengths were
fixed at 280 and 410 nm, respectively and the slit width was set at 10/10 nm.
2.5. Use of circular dichroism spectroscopy to study the interactions between FMFS NPs
and BSA
The circular dichroism (CD) spectroscopy is often used to study the binding interactions between
a biomolecule (like protein) and a ligand. The secondary structure of a protein may be changed
during its interactions with a ligand and this change of protein structure gives rise to a
characteristic CD spectrum [34].
8

For the present case, the CD experiments were conducted in the wavelength range of 190-250
nm on CD spectropolarimeter at room temperature under dry N2 atmosphere. The scan speed was
fixed at 100 nm/min with a band width of 10 nm. The scans were accumulated and averaged
automatically by the instrument. All experiments were performed in Tris-HCl buffer (10 mM,
pH 7.4) at 25oC. During the CD studies, the concentration of BSA was kept constant at 9 μM,
while the concentration of FMFS NP solution was varied. The α-helical content of BSA was
calculated according to the following equation [29]:
-MRE 208- 4000
33, 000- 4000
………….. (1)
α-helix (%) =
X 100
Here, MRE208 is the observed mean residue ellipticity (MRE) value at 208 nm. The MRE of a
pure α-helix at 208 nm is 33,000 and that of β-form and random coil conformation at 208 nm is
4000. The MRE value can be calculated by the following equation [29]:
Observed CD (m deg)
(Cpnl ×10)
………………. (2)
MRE =
Here, Cp is the molar concentration of BSA, n is the number of the amino acid residues (583 for
BSA) and l is the path length (0.1 cm) [29].
3. Results and discussion
3.1. Surface structure and morphology of the FMFS NPs
The present functionalization process induced significant changes in the surface structure,
morphology and spectroscopic behavior of the FMFS NPs as compared to those of the bare
silica NPs.
Very often, FT-IR spectroscopy is found to be helpful in understanding the organic
functionalities adsorbed on the NP surface and their binding interaction with NP surface. In the
present case, extensive IR spectroscopic studies of all possible systems involved in the synthesis
of FMFS NPs (including the bare silica NPs also) were done and the results are shown in Fig. 2
(a)(i-v). The distinctive features of each spectrum presented in this figure confirm the surface
functionalization of silica NPs through APTES mediated grafting of (3) onto the NP surface via
sulfonamide bond. In its free form, (3) showed IR stretching frequencies in the range of 1182 and
1339-1370 cm-1 [Fig. 2(a)(iv)] which are associated with its S=O (SO3H) functionality. Both of
9

these two peaks were found to be broadened and shifted after the attachment of (3) with the
APTES modified silica (APTES-silica) NPs [Fig. 2(a)(v)]. The N-H (primary amine) stretching
frequency of APTES-silica NPs appeared in the range of 2865-2930 cm-1 [Fig. 2(a)(ii)] but this
was found to be shifted to the range of 2940-3052 cm-1 in case of FMFS NPs [Fig. 2(a)(v)]. At
the same time, broadening of the peak was also observed [Fig. 2(a)(v)]. This IR data may be
associated with the -N(H)-SO2- functionality present onto the surface of FMFS NPs. These
results confirmed the attachment of the fluorescent flavone analog (3) onto the surface of FMFS
NPs through sulfonamide bond formation [39].
Fig. 2(b) shows the DLS size distribution plots of bare silica NPs and FMFS NPs. The mean
particle size (polydispersity index or PDI) of silica NPs and FMFS NPs were found to be 238.8
nm (PDI 0.325) and 443.7 nm (PDI 1) respectively. The zeta potential values of bare silica and
FMFS NPs were found to be -15.0 mV and 10.2 mV respectively, at pH 7.4. The decrease in
negative value of the zeta potential of FMFS NPs can be attributed to the fact that the negative
charges of the APTES-silica system were partially neutralized due to its functionalization with
flavone moiety (3) [33].
Figure 2
The surface study of FMFS NPs was done by TGA analysis to further confirm the presence of
flavone moiety at the NP surface. For comparison, TGA of each of the components involved in
the synthesis of FMFS NPs were also done.
The TGA experiments were carried out in the temperature range of 40-850oC under N2
atmosphere at a heating rate of 10oC min-1. For all the samples, an initial weight loss was
observed up to 100oC. This may be due to the presence of moisture in the sample and
characteristic TGA plots for these samples were obtained in the temperature range of 100-850oC.
The results are shown in Fig. 3 [(a) (i-iii)]. The weight losses of various systems involved were
found to be: 8.68% for silica NPs (i), 19.49% for APTES-silica (ii) and 31.55% for FMFS NPs
(iii) [Fig. 3 [(a) (i-iii)]. The surface concentration of (3) was calculated from TGA data and it
was found to be 571.57µM g-1. TGA plots [Fig. 3 [(a) (i-iii)] indicated higher thermal stability of
the FMFS NPs as compared to the amine functionalized silica NPs (i.e. APTES-silica).
The specific surface area (SSA) of FMFS NPs was measured by BET (Brunauer Emmett Teller)
analysis and it was found to be 68 m2 g-1. The concentration of (3) loaded on the NP surface was
calculated as 8.405 µM m-2 [Fig. 3(b)]. The corresponding pore size distribution plots are shown
10

in the inset of Fig. 3 (b). This was estimated by using the NLDFT (non-local density functional
theory) model. The pore dimension for FMFS NPs was found to be 2.55 nm.
Figure 3
Figs. 4a (i) and (ii) show the HR-TEM images of silica NPs before and after their surface
functionalization respectively. The corresponding close-up views are shown in the insets of these
figures. TEM images confirmed that in both the cases, NPs were spherical. So the present
functionalization method did not alter the grooved structure of these NPs. The color of the
FMFS NP solution under visible and UV light (λex 365 nm) are shown in Figs. 4(b) (iv) and (v),
respectively. It is evident that the color of the FMFS NPs originated from the color of the
precursor flavone analog, (2) [Fig. 4(b)(i) and (ii)]. Moreover, the fluorescence behavior of
flavone (2) [Fig. 4(b)(iv)] was also responsible for the origin of the intrinsic fluorescence activity
of the FMFS NPs [Fig. 4(b)(v)].
Figure 4
3.2. Interactions of FMFS NPs with BSA
3.2.1. Quenching of fluorescence of FMFS NPs in presence of BSA
Silica NPs functionalized with organic fluorophore have been explored as biomolecule sensors in
the biological and medical diagnostics. Characteristic changes of spectroscopic behavior of these
NPs are observed during their interaction with biomolecules, like proteins and nucleic acids [40-
42]. In this type of study, BSA is usually selected as model (protein) biomolecule due to its easy
availability, low cost, aqueous solubility, characteristic ligand binding affinity and intrinsic
fluorescence activity associated with its two tryptophan residues, Trp-212 and Trp-134. These are
located at the hydrophobic binding sites and on the surface of the BSA protein, respectively [40-
42].
The absorption spectra of the FMFS NPs in the presence and absence of BSA are shown in Fig.
5(a). A broad but strong absorption band was observed at ~ 420 nm with a shoulder at ~355 nm.
These bands may correspond to the band I and II of the flavone moiety [18]. The strong
absorption band of FMFS NPs solution at ~ 420 nm in the absence of BSA may be due to the π-
π* transition [27, 43] of the flavone moiety, present on the FMFS NPs surface. Upon addition of
BSA, a prominent peak shift (both in terms of peak intensity and max value) of the FMFS NPs
solution was observed [Fig. 5(a)]. As the concentration of BSA gradually increased (2-10 µM),
the absorbance at max 355 nm was found to be decreased. At the same time, the absorbance at
11

420 nm increased with the appearance of an isobestic point at 380 nm [Fig. 5(a)]. These changes
reflected the formation of 1:1 complex between FMFS NPs and BSA [44].
The fluorescence emission of the FMFS NPs in the absence and presence of different
concentration of BSA are shown in Fig. 5(b). In absence of BSA, the typical dual emission bands
of the flavone analog were observed at max ~ 410 nm and 495 nm (broad shoulder) respectively
[27,30]. Hence, it is clear that the flavone moieties were present on the surface of FMFS NPs
and they imparted their fluorescence characteristics to the NP system whereby fluorescent silica
(FMFS) NPs were obtained. Upon gradual addition of BSA (0.3-4.8 µM), the fluorescence
spectrum of FMFS NPs was found to be changed [Fig. 5(b)]. The emission band at em 410 nm
was found to be gradually quenched on addition of BSA solution, whereas the small hump at em
495 nm gradually built up with time. The same phenomenon was observed in the case of the
quenching of intrinsic fluorescence of BSA in presence of FMFS NP solutions of various
concentrations [Fig. 6(a)]. The fluorescence emission intensity of FMFS NPs at em 495 nm was
found to be ~6 times enhanced when concentration of BSA was 5 µM. The appearance of
emission peak along with enhanced fluorescence intensity (at longer wavelength) can be
rationalized on the basis of the binding interactions of FMFS NPs with the appropriate
functionalities present at the hydrophilic site of BSA. This type of binding interaction leads to
the formation of new complex which showed characteristic fluorescence activity [44].
The present binding interactions of FMFS NPs with BSA were studied spectrophotometrically in
aqueous medium. Hence, this functionalization method of silica NPs evidently enhanced the
hydrophilicity of the hydrophobic flavone moiety e.g. (2) after its attachment onto the NP
surface.
The fluorescence anisotropy measures the rotational diffusion property of a molecule. When a
reactant binds to a substrate and forms a complex which is larger in size with respect to both the
reactant and the substrate, a lower rotational diffusion coefficient and higher fluorescence
anisotropy (r) are observed. Thus, the restriction imposed by BSA on FMFS NPs can be better
understood by anisotropy measurements. The anisotropy of FMFS NPs solution was plotted
against the concentration of BSA solution (6-22.8 µM) [Fig. 5(c)]. The increase in anisotropy
with the increase in BSA concentration reflected the fact that the FMFS NPs experienced more
constrained environment during their binding with BSA [38].
12

The binding constant values were estimated from the corresponding fluorescence emission data
by using Benesi-Hildebrand equation (3) [40, 44]:
1
1
1
……………… (3)
=
+
[I1 − I0] K [BSA]
[I−I0]
[I1 −I0]
In the above equation, I0 and I are the emission intensities of the FMFS NPs solution in the
absence and presence of BSA, respectively. I1 is the emission intensity of FMFS NP-BSA
complex. The emission intensity at em 495 nm was used to derive the Benesi-Hildebrand plot
for binding interaction of FMFS with BSA [Fig. 5(d)]. A good linear correlation (r2 = 0.99) was
found for the 1/ (I−I0) versus 1/ [BSA] plot. This confirmed again the formation of 1:1 inclusion
complex between FMFS NPs and BSA. The binding constant for FMFS NPs was calculated as
7.90×104 M−1. Over a range of BSA concentrations, the fluorescence intensities of the FMFS NP
solution showed good linear dependence [r2 = 0.99] as shown in the inset of Fig. 5(d)].
Figure 5
It was really interesting to note that not only the fluorescence activity of the FMFS NPs was
found to be changed but the fluorescent behavior of BSA was also highly influenced by FMFS
NPs. Thus, the sensing of biomolecules like BSA by this type of hybrid nanomaterials can be
monitored by tracking either the change of fluorescent activities of FMFS NPs or BSA.
3.2.2. Quenching of fluorescence of BSA during its interaction with FMFS NPs
BSA exhibited strong fluorescence intensity at λem of 345 nm which can be attributed to its
tryptophan residues [29,44]. The intrinsic fluorescence of BSA was found to be quenched
significantly in the presence of FMFS NPs. The continuous quenching of fluorescence of BSA
was observed with the increase of concentration of FMFS NP solution. On the other hand, a
new fluorescence emission peak was noticed at λem 495 nm and a continuous growth of this
emission band was quite visible accompanying a gradual red shift [Fig. 6(a)]. During blank
experiments, FMFS NPs showed no emission at wavelength of 345 nm when excited at the same
wavelength. Thus, it can be inferred that the intrinsic fluorescence of BSA was quenched by
Förster resonance energy transfer (FRET) mechanism. The intensity of fluorescence emission
peak at λem 410 nm (which is the characteristic of FMFS NP itself) was also found to be
increased on gradual addition of FMFS NP solution [Fig. 6(a)].
The fluorescence activity of the FMFS NPs was due to the fluorescent organic functionalities
attached onto the surface of these NPs and under the hydrophobic environment of BSA protein,
13

this fluorescence activity was found to be enhanced. It is interesting to note that the new
fluorescence emission peak at em 495 nm did not resemble to either the fluorescence emission
peak of BSA or FMFS NPs. Moreover, the appearance of an isobestic point at wavelength value
of 382 nm indicated the presence of both BSA and FMFS NPs species at equilibrium. All these
results clearly indicated the formation of BSA-FMFS NP complex for which the new
fluorescence emission peak was observed. The quenching of fluorescence of the tryptophan
residue of BSA may be elaborated with the help of Stern-Volmer equation (4) [43]:
F0
= 1+ kqτ0 [Q] = 1 + KSV [Q]
…………….. (4)
F
Where kq is the bimolecular quenching rate constant, KSV is the Stern-Volmer constant, τ0 is the
fluorescence lifetime of the protein in the absence of quencher (i.e. FMFS NPs in the present
case). [Q] denotes the concentration of FMFS NPs. The linear Stern-Volmer plot was obtained
with increasing concentration of NPs [Fig. 6(c)] suggesting the occurrence of static fluorescence
quenching of BSA during the binding process. The Stern-Volmer quenching constant (KSV) was
calculated from the slope of Stern-Volmer plot and the value was found to be 1.91±0.014×105 M-
1 [Fig. 6(c)]. The value of the bimolecular quenching constant (kq) was obtained as 3.81×105 M-
1s-1. It was found to be greater than the limiting (highest possible) value of 1×1010 M-1s-1 in
aqueous medium [15]. The excitation spectrum of BSA was found to be quenched in the
presence of FMFS NPs [Fig. 6(b)]. This is a clear evidence of the occurrence of specific types of
interactions between BSA and FMFS NPs [44]. From the UV-Vis studies, it can be seen that the
UV–Vis spectra of BSA did not overlap with the BSA-FMFS complex spectra [Fig. 6(d)]. This
indicated the possibility of the occurrence of a ground state complexation process.
Figure 6
3.2.3. Determination of the binding constant and the number of binding sites for the
interaction between FMFS NPs and BSA
From the linearity of the Stern-Volmer plot with increasing concentration of FMFS NPs, static
quenching interactions between BSA and FMFS NPs could be inferred. The binding constant
(Kb) and the number of binding sites (n) can be determined according to the method described by
Jiang et al. using the following equation [29]:
log (F0 –F)
……………… (5)
log Kb + n log [Q]
F
14

Here, Kb is the binding constant for the interaction of FMFS NPs with BSA and n is the number
of binding sites. This can be determined from the intercept and the slope of the double logarithm
regression curve of log (F0-F/F) versus log [Q] based on the Eq. (5) [Fig. 7 (a)]. The value of Kb
was found to be 2.36±0.016×105 M-1. The value of n was obtained ~1 which indicated the
presence of single binding site in BSA for the FMFS NPs.
3.2.4. Circular dichroism measurements of binding interaction between FMFS NPs and
BSA
The CD spectrum of BSA exhibited two negative bands in the UV region of 208 nm (π→π*
transition) and 222 nm (n→π* transition) as shown in Fig. 7(b). This is the characteristic feature
of the α-helical structure of a protein. On interaction of FMFS NPs with BSA, the CD intensities
of the BSA bands at all wavelengths (in the far UV region) were found to be decreased.
However, any noticeable change in the peak position was not observed. The α-helical content of
free BSA was 53.5% and it decreased to 51.7, 45.8, 37.8 and 25.3%, respectively upon binding
with FMFS NPs at 1:2, 1:4, 1:6 and 1:8 molar ratios. Thus, it can be concluded that some
conformational changes in BSA protein structure occurred on binding with this fluorescent NP
system. The secondary structure and helicity of the BSA protein was found to be retained as the
CD spectra remained similar in shape after its binding with FMFS NPs system [29].
Figure 7
3.3. Antioxidant activity of FMFS NPs in aqueous medium
One of the major problems associated with small molecule based antioxidants like curcumin and
flavonoids is their poor water solubility and hence low bioavailability in aqueous medium. In the
present work, a flavone moiety was tagged onto the surface of the silica NPs (FMFS NPs) and
the antioxidant activity of these FMFS NPs was determined in aqueous medium by ferric
reducing antioxidant potential assay method. Results are shown in Fig. S1 (Supporting
Information, page S9).
In general, this method does not give good results for flavone moiety due to its hydrophobicity.
In the present case, after its attachment on APTES-silica surface, hydrophilicity of the flavone
moiety was found to be enhanced. It was observed that the FMFS NPs responded positively to
this test and showed a ferric reducing antioxidant potential of 55.6 ± 0.06 mM of Gallic acid
equivalent (GAE) g-1. This value is comparable to that of the well-known naturally occurring
15

antioxidants, like quercetin [45]. Hence, the FMFS NPs may have the potential to carry the
hydrophobic antioxidant in the interior of the aqueous system similar to biological cells.
As discussed in the previous section, the strong and intrinsic fluorescence activity of FMFS NPs
was originated due to its attachment with the fluorescent flavones moiety and this fluorescent
activity was drastically influenced by its interaction with biomolecule like, BSA. All these
characteristics make it an ideal candidate for biomedical applications like sensing biomolecules
in aqueous system. The change of intrinsic fluorescence activity of FMFS NPs during their
interaction with BSA may help to visualize these biomolecules. However, for the use of any
probe or sensor in the detection of biomolecules related to living system, one of the essential
conditions is its biocompatibility. Hence, in the present case we have tested the (%) hemolysis
activity and cell viability of FMFS NP to get an idea about their biocompatibility.
3.4. Biocompatibility of the FMFS NPs
Hemolysis tests were carried out in vitro to ascertain the safety of the FMFS NPs system
towards human red blood cells. Fig. 8(a) shows the (%) hemolysis of silica NPs before the
functionalization (i.e. bare silica NPs) and FMFS NPs where the fluorescent flavone analog was
attached to the surface of the silica NPs. Three different concentrations (0.25, 0.5 and 1 mg ml-1)
were taken for all the samples (except water) to compare the degree of damage to red blood cells
with respect to distilled water. In comparison to bare silica NPs, the FMFS NPs showed less
hemolysis. From the figure [Fig. 8(a)], it is evident that the attachment of the synthetic flavone
analog on the silica nanoparticle surface increased its blood compatibility.
The cytotoxicity of FMFS NPs along with flavone (2) was tested by the MTT assay to evaluate
the tolerance of cells in the presence of these systems at their working concentrations. The cells
were incubated with different concentrations (2, 5, 10, 20, 50, 100 µM) of (2) and FMFS NPs
for 24 hour. The results show that the cell viabilities were close to 90% at 20µM dose. Thus, the
working concentrations of (2) and FMFS NPs did not cause any cell apoptosis.
It is interesting to note that, these non-toxic FMFS NPs showed strong fluorescence activity
under red (λex 587/25 nm), blue (λex 365 nm) and green (λex 450-490 nm) channels in cellular
environment [Fig. 8(d1-4)] but the original hydrophobic flavone moiety [i.e. flavone (2)] without
its attachment with APTES-modified silica NPs did not show fluorescence activity in the cellular
medium [Fig. 8(c1-4)]. So FMFS NPs are eventually an ideal candidate for probing biomolecules,
like BSA in aqueous system.
16

Figure 8
4. Conclusion
In the present study for the first time, a fluorescent synthetic antioxidant flavone analog was
attached on the surface of APTES modified mesoporous silica NP matrix to synthesize flavone
modified fluorescent silica (FMFS) NPs. These NPs were then subjected to full characterization
in terms of their surface chemistry and morphology. Synthesized fluorescent NPs offered several
new advantages. On the one hand, they were found to enhance the bioavailability of the flavone
moiety in the aqueous system. On the other hand, the surface attached flavone moiety imparted
characteristic fluorophoric and antioxidant property to the FMFS NPs, making them more
biocompatible in comparison with modified fluorescent silica NPs employing synthetic
fluorophores.
Their characteristic fluorescence, along with their hydrophilicity and biocompatibility (in terms
of the blood and cell viability along with antioxidant activity in aqueous medium), have ensured
that these NPs have the potential to be used as biomolecule sensors. In addition, the fact that
these FMFS NPs retain their fluorescence properties even inside the cellular medium implies
that they are highly suited to perform as fluorescence probes for cellular biomolecules.
As part of our future plans, we aim to incorporate many similar such flavone fluorophores onto
the silica NPs surface using the present strategy or variations thereof, and examine the
performance of the resultant FMFS NPs as biomolecule sensors and live cell image contrast
agents. Since the fluorescence and physico-chemical properties of flavones are extremely
sensitive to the nature and position of ring substituents, we expect to build up in this way, an
exhaustive library of FMFS NPs with an immense window of functional properties relevant to
biological and medical diagnostics.
Acknowledgements
We thank SERB-DST [sanction order no. SR/SO/BB-0007/2011 dated 21.08.2012 to N. A. B.]
for the financial support. S. D., S.B. and M.N. A. thank CSIR [Sanction No. 01 (2504)/11/EMR-
II) to N.A.B.], MANF-UGC and SERB-DST [sanction order no. SR/SO/BB-0007/2011 dated
21.08.2012 to N. A. B.] respectively for their fellowships. We thank the Dept. of Chemistry,
Siksha Bhavana, Visva Bharati University and its DST-FIST and UGC-SAP (Phase-II)
programme for necessary infrastructural and instrumental facilities. We acknowledge Prof. T.
Basu, Department of Bio-chemistry and Bio-physics, University of Kalyani, Kalyani-741235,
17

W.B., India for his kind help in DLS measurements. The acknowledgement is also due to the
Centre for Research in Nanoscience & Nanotechnology, University of Calcutta, India for the
TEM facility.
A. Supplementary Information
Supplementary information [General chemicals used and instrumentation, experimental
procedures for the synthesis of the flavone analog, spectroscopic data for the confirmation of its
structure, procedure of ferric reducing antioxidant potential assay, in-vitro erythrocyte induced
blood viability assay and MTT assay, Method of fluorescence cell imaging by (2) and FMFS
NPs] associated with this article can be found, in the online version.
18

References
[1] A. Liberman, N. Mendez, W. C. Trogler, A. C. Kummel, Synthesis and surface
functionalization of silica nanoparticles for nanomedicine, Surf. Sci. Rep. 69 (2014) 132-158.
[2] B. Hazer, E. Akyol, Efficiency of gold nano particles on the autoxidized soybean oil
polymer: fractionation and structural analysis, J Am. Oil Chem. Soc. 93 (2016) 201-213.
[3] L. Wang, W. Zhao, W. Tan, Bioconjugated silica nanoparticles: Development and
applications, Nano Res. 1 (2008) 99-115.
[4] K. Chitra, G.Annadurai, Fluorescent silica nanoparticles in the detection and control of the
growth of pathogen, J. Nanotech. 2013 (2013) 1-7.
[5] J. Qian, X. Li, M. Wei, X. Gao, Z. Xu, S. He, Bio-molecule-conjugated fluorescent
organically modified silica nanoparticles as optical probes for cancer cell imaging, Opt. Express
16 (2008) 19568-19578.
[6] P. C. Chen, S. C. Mwakwari, A. K. Oyelere, Gold nanoparticles: From nanomedicine to
nanosensing, Nanotech. Sci. Appl. 1 (2008) 45-66.
[7] O. Masala, R. Seshadri, Synthesis routes for large volumes of nanoparticles, Annu. Rev.
Mater. Res. 34 (2004) 41-81.
[8] B. Kumar, P. R. Yadav, H. C. Goel, M. M. A. Rizvi, Recent developments in cancer therapy
by the use of nanotechnology, Dig. J. Nanomater. Biostruct. 4 (2009) 51–57.
[9] M. J. Thompson, D. Bashford, L. Noodleman, E.D. Getzoff, Photoisomerization and proton
transfer in photoactive yellow protein, J. Am. Chem. Soc. 125 (2003) 8186-8194.
[10] F. L. Yen, T. H. Wu, C. W. Tzeng, L. T. Lin, C. C. Lin, Curcumin nanoparticles improve
the physicochemical properties of curcumin and effectively enhance its antioxidant and
antihepatoma activities, J. Agric. Food Chem. 58 (2010) 7376-7382.
[11] T. Mokari, S. E. Habas, M. Zhang, P. Yang, Synthesis of lead chalcogenide alloy and core-
shell nanowires, Angew. Chem. Int. Ed. 47 (2008) 5605-5608.
[12] S. Panigrahi, S. Praharaj, S. Basu, S. K. Ghosh, S. Jana, S. Pande, T. Vo-Dinh, H. Jiang, T.
Pal, Self-assembly of silver nanoparticles: Synthesis, stabilization, optical properties, and
application in surface-enhanced Raman scattering, J. Phys. Chem. B 110 (2006) 13436-13444.
[13] V. M. Rotello, Nanoparticles: Building Blocks for Nanotechnology, Kluwer Academic
Publishers, New York, USA, 2004.
19

[14] H. Wanyika, E. Gatebe, P. Kioni, Z. Tang, Y. Gao, Synthesis and characterization of
ordered mesoporous silica nanoparticles with tunable physical properties by varying molar
composition of reagents, Afr. J. Pharm. Pharmacol. 5 (2011) 2402–2410.
[15] H. A. Benesi, J. H. Hildebrand, A spectrophotometric investigation of the interaction of
iodine with aromatic hydrocarbons. J. Am. Chem. Soc. 71 (1949) 2703–2707.
[16] G. Chen, F. Song, X. Wang, S. Sun, J. Fan, X. Peng, Bright and stable Cy3-encapsulated
fluorescent silica nanoparticles with a large strokes shift, Dyes pigm. 93 (2012) 1532-1537.
[17] P. K. Sengupta, M. Kasha, Excited state proton-transfer spectroscopy of 3-
hydroxyflavone and quercetin, Chem. Phys. Lett. 68 (1979) 382-385.
[18] S. Mondal, S. Basu, D. Mandal, Ground-and excited-state proton-transfer reaction of 3-
hydroxyflavone in aqueous micelles, Chem. Phys. Lett. 479 (2009) 218-223.
[19] N.A. Begum, N. Roy, S. Mandal, S. Basu, D. Mandal, Fluorescence spectroscopy of a
naturally occurring carbazole alkaloid: murrayanine, J. Lumin. 129 (2) (2009) 158-163.
[20] T. A. Geissman, (ed.), The chemistry of flavonoid compounds, Pergamon Press, Oxford,
1962.
[21] J. B. Harborne, T. J. Mabry, The flavonoids: Advances in research, Chapman and Hall,
London, 1982.
[22] T. J. Murray, X. Yang, D. H. Sherr, Growth of a human mammary tumor cell line is
blocked by galangin, a naturally occurring bioflavonoid, and is accompanied by down-
regulation of cyclins D3, E, and A, Breast Cancer Res. 8 (2006)1-12.
[23] S. Das, I. Mitra, S. Batuta, M. N. Alam, K. Roy, N.A. Begum, Design, synthesis and
exploring the quantitative structure-activity relationship of some antioxidant flavonoid analogs,
Bioorg. Med. Chem. Lett. 24 (2014) 5050-5054.
[24] N. Desideri, I. Sestili, M. L. Stein, E. Tramontano, S. Corrias, P. L. Colla, Synthesis and
anti-human immunodeficiency virus type 1 integrase activity of hydroxybenzoic and
hydroxycinnamic acid flavon-3-yl esters, Antiviral Chem. Chemother. 9 (1998) 497-509.
[25] S. Alam, S. Mostahar, Studies of antimicrobial activity of two synthetic 2',4',6'-
trioxygenated flavones, J. Applied Sci. 5 (2005) 327-333.
[26] N. A. Begum, N. Roy, R. A. Laskar, K. Roy, Mosquito larvicidal studies of some chalcone
analogs and their derived products: structure-activity relationship analysis, Med. Chem. Res. 20
(2011) 184-191.
20

[27] (a) D. Ghosh, S. Batuta, S. Das, N. A. Begum, D. Mandal, Proton Transfer Dynamics of
4′-N, N-Dimethylamino-3-hydroxyflavone observed in hydrogen-bonding solvents and aqueous
micelles, J. Phys. Chem. B 119 (17) (2015) 5650-5661. (b) D. Ghosh, A. K. Pradhan, S.
Mondal, N. A. Begum, D. Mandal, Proton transfer reactions of 4′-chloro substituted 3-
hydroxyflavone in solvents and aqueous micelle solutions. Phys. Chem. Chem. Phys. 2014, 16
(18), 8594-8607. (c) V. G. Pivovarenko, A. Wróblewskab, J. Błażejowski, 2-[4-
(Dimethylamino) phenyl]-3-hydroxy-4H-chromene-4-one: A H-bond-sensitive fluorescent
probe for investigating binary mixtures of organic solvents, Anal. Chim. Act. 545 (2005)74-78.
[28] H. Pool, S. Mendoza, H. Xiao, D. J. McClements, Encapsulation and release of
hydrophobic bioactive components in nanoemulsion-based delivery systems: impact of physical
form on quercetin bioaccessibility, Food Funct. 4 (2013) 162-174.
[29] (a) A. Singha Roy, D.R. Tripathy, A. Chatterjee, S. Dasgupta, A Spectroscopic study of the
interaction of the antioxidant naringin with bovine serum albumin, J. Biophys. Chem. 1 (2010)
141-152. (b) N. Shahabadi, M. Mohammadpour, Study on the interaction of sodium morin-5-
sulfonate with bovine serum albumin by spectroscopic techniques, Spectrochim. Acta Mol.
Biomol. Spectrosc. 86 (2012) 191-195.
[30] D. Ghosh, G. Ahamed, S. Batuta, N. A. Begum, D. Mandal, Effect of an electron-donating
substituent at the 3', 4'-position of 3-Hydroxyflavone: photophysics in bulk Solvents, J. Phys.
Chem. A 120 (2016) 44-54.
[31] A. M. G. Silva, A. C. Tomé, A. M. S. Silva, J. A. S. Cavaleiro, Chlorosulfonation of
flavones, Phosphorus, Sulfur and silicon. 140 (1998) 113-124.
[32] P. Stathi, M. Louloudi, Y. Deligiannakis, EPR study of phenolic radical stabilization by
grafting on SiO2, Chem. Phys. Lett. 472 (2009) 85-89.
[33] Y. Deligiannakis, G.A. Sotiriou, S.E. Pratsinis, Antioxidant and antiradical
SiO2 nanoparticles covalently functionalized with gallic acid, ACS Appl. Mater. Interfaces 4
(2012) 6609-6617.
[34] K. C. Christoforidis, M. Louloudi, A. W. Rutherford, Y. Deligiannakis, Semiquinone in
molecularly imprinted hybrid amino acid-SiO2 biomimetic materials. An experimental and
theoretical study, J. Phys. Chem. C 112 (2008) 12841-12852.
21

[35] L. K. Koopal, Y. Yang, A. J. Minnard, P. L. M. Theunissen, W. H. Riemsdijk, Chemical
immobilisation of humic acid on silica, Colloid Surf. A: Physicochem. Eng. Aspects 141 (1998)
385-395.
[36] (a) M. Oyaizu, Studies on products of browning reactions: Antioxidative activities of
products of browning reaction prepared from glucosamine, Jpn. J. Nutr. 44 (1986) 307-315. (b)
R. A. Laskar, I. Sk, N. Roy, N. A. Begum, Antioxidant activity of Indian propolis and its
chemical constituents, Food Chem. 122 (2010) 233-237.
[37] N. R. Ravikumara, B. Madhusudhan, T. S. Nagaraj, S. R. Hiremat, G. Raina, Preparation
and evaluation of nimesulide-loaded ethylcellulose and methylcellulose nanoparticles and
microparticles for oral delivery, J. Biomater. Appl. 24 (2009) 47-64.
[38] A. Singha Roy, A. K. Dinda, S. Chaudhury, S. Dasgupta, Binding of antioxidant flavonol
morin to the native state of bovine serum albumin: Effects of urea and metal ions on the
binding, J. Lumin. 145 (2014) 741-751.
[39] R. M. Silverstein, F. X. Webster, D. J. Kiemle, D. L. Bryce, Spectrometric Identification
of Organic Compounds, 8th ed., Wiley, USA, 2014.
[40] M. Asha Jhonsi, A. Kathiravan, R. Renganathan, Spectroscopic studies on the interaction of
colloidal capped CdS nanoparticles with bovine serum albumin, Colloids Surf. B Biointerfaces
72 (2009) 167-172.
[41] M. Dockal, D.C. Carter, F. Rüker, Conformational transitions of the three recombinant
domains of human serum albumin depending on pH, J. Biol. Chem. 275 (2000) 3042–3050.
[42] R.E. Olson, D.D. Christ, Plasma protein binding of drugs, Ann. Rep. Med. Chem. 31
(1996) 327-336.
[43] S. Zhanga, X. Suna, Z. Jing, F. Qua, Spectroscopic analysis on the resveratrol-DNA
binding interactions at physiological pH, Spectrochim. Acta Mol. Biomol. Spectrosc, 82 (2011)
213-216.
[44] (a) N. Dash, A. Mishra, G. Krishnamoorthy, Alkyl chain dependent interactions of ligands
with bovine serum albumin, J. Pharm. Biomed. Anal. 77 (2013) 55-62. (b) J. R. Lakowicz,
Principles of Fluorescence Spectroscopy, 3rd ed., Springer, New York, USA, 2006.
[45] O. Firuzi, A. Lacanna, R. Petrucci, G. Marrosu, L. Saso, Evaluation of the antioxidant
activity of flavonoids by "ferric reducing antioxidant power" assay and cyclic voltammetry,
Biochim. Biophys. Acta, 1721 (2005) 174-84.
22

Figure legends
Fig. 1. Structures of the parent flavone skeleton (1), 3-hydroxy-4-methoxyflavone (2) and 3,4'-
dimethoxy-3'-flavone sulfonic acid (3).
Fig. 2. (a) FT-IR spectra of (i) silica nanoparticles before their functionalization, (ii) APTES
itself, (iii) APTES-silica nanoparticle system, (iv) 3,4-dimethoxy-3-flavone sulfonic acid (3),
(v) flavone modified fluorescent silica (FMFS) nanoparticles (b) Dynamic light scattering (DLS)
size distributions of silica nanoparticles (i) and FMFS nanoparticles (ii).
Fig. 3. (a) Thermo gravimetric analysis (TGA) of (i) bare silica nanoparticles and (ii) APTES-
silica nanoparticles, (iii) flavone modified fluorescent silica (FMFS) nanoparticles (b) specific
surface area study of FMFS nanoparticles by N2 adsorption ( ) and desorption ( ) isotherms at
77 K. Corresponding NLDFT (non-local density functional theory) pore size distributions are
shown in the inset.
Fig. 4. (a) TEM images of (i) silica nanoparticles before their functionalization and (ii) flavone
modified fluorescent silica (FMFS) nanoparticles. Insets of these figures show the close-up view
(b) Color of (i) silica nanoparticles before functionalization, (ii) 3-hydroxy-4-methoxyflavone
(2) itself and (iii) FMFS nanoparticles under visible light. Color of (iv) 3-hydroxy-4-
methoxyflavone (2) and (v) FMFS nanoparticles when illuminated with UV light (λex 365 nm).
Fig. 5. (a) Absorption and (b) fluorescence emission spectra of flavone modified fluorescent
silica (FMFS) nanoparticles solution (20µM) in absence and presence of BSA (c) Fluorescence
anisotropy(r) of FMFS nanoparticles solution (20 μM) in presence of different concentrations (6-
22.8 μM) of BSA (d) Benesi-Hildebrand plot for the interaction of FMFS nanoparticles (20 µM)
with BSA (0.3-4.8 µM). The inset of (d) shows the variations of fluorescence intensities of
FMFS nanoparticles solution (at λmax 495 nm) as a function of BSA concentration.
Three replicates were performed for each and every experiment. Results are expressed as
mean ± SD with error bars indicating SD (SD=standard deviations of three independent
experiments).
23

Fig. 6. (a) Fluorescence emission spectra of BSA (2 µM) in the presence of different
concentrations of flavone modified fluorescent silica (FMFS) nanoparticle solution (2-32 µM)
(b) Corresponding fluorescence excitation spectra (c) Stern-Volmer plot for the interaction of
FMFS nanoparticles (2-32 µM) with BSA (2 µM) at λex=280 nm (d) UV-Vis spectra of BSA and
BSA-FMFS nanoparticle complex.
Three replicates were performed for each and every experiment. Results are expressed as
mean ± SD with error bars indicating SD (SD=standard deviations of three independent
experiments).
Fig. 7. (a) Calculation of the binding constants for the interaction of flavone modified
fluorescent silica (FMFS) nanoparticles (2-32µM) with BSA (2 µM) (b) Circular dichroism
(CD) spectra of the BSA before and after its binding with FMFS nanoparticles. CD spectra of
five samples having BSA and FMFS in the molar ratios of 1:0, 1:2, 1:4, 1:6 and 1:8 were
recorded in 10 mM Tris-HCl buffer of pH 7.4.
Three replicates were performed for each and every experiment. Results are expressed as
mean ± SD with error bars indicating SD (SD=standard deviations of three independent
experiments).
Fig. 8. (a) % hemolysis activity of bare silica and FMFS nanoparticle samples. DW (distilled
water) was used as positive control. Inset shows corresponding reduced plot. (b) Cytotoxicity
study with 3-hydroxy-4-methoxyflavone (2) and FMFS nanoparticle samples having various
concentrations (0, 2, 5, 10, 20, 50, 100µM) in human normal lung (L132) cell for 24 hour.
Fluorescence images of human lung cancer (A549) cell incubated with (2) (c1-4) and FMFS
nanoparticles (d1-4) for 30 min in bright field (c1 and d1) and under exposure of fluorescence
light using a green (c2 and d2), blue (c3 and d3) and red (c4 and d4) filter. Magnification - 40X.
Three replicates were performed for each and every experiment. Results are expressed as
mean ± SD with error bars indicating SD (SD=standard deviations of three independent
experiments)
Scheme 1. Synthesis of 3-hydroxy-4-methoxyflavone (2) and its analog, 3,4'-dimethoxy-3'-
flavone sulfonic acid (3).
24

Scheme 2. Surface functionalization of APTES-modified silica nanoparticles with sulfonic acid
analog of flavone (3). Reagents and condition: (i) Toluene, 72 hr Reflux; (ii) Et3N, toluene, 12
hr, reflux.
.
25

(1)
(2)
(3)
Fig. 1.
26

50
40
30
20
10
(a)
ii
v
iv
iii
i
ii
(b)
i
500
1000
1500
2000
2500
3000
3500
0
500
1000
1500
2000
2500
3000
Wavenumber (Cm-1)
Size (d.nm)
Fig. 2.
27

60
50
40
30
20
10
100
95
90
85
80
75
70
65
0.0025
0.0020
0.0015
0.0010
0.0005
0.0000
Pore Volume 0.0692 ccg-1
NLDFT Model
(a)
(b)
2.55 nm
i
0
10
20
30
40
50
Pore width (nm)
Adsorption
Desorption
ii
Surface area 68 m2g-1
iii
0.0
0.2
0.4
0.6
0.8
1.0
200
400
600
800
Relative pressure [P/P0]
Temparature (oC)
Fig. 3.
28

(v)
(i)
(ii)
(iii)
(iv)
(i)
(ii)
(a)
(b)
Fig. 4.
29