Advanced Multifunctional Inorganic Nanostructured Oxides for Controlled Release and Sensing (Soumit S. Mandal)
Akreditasi LIPI Nomor : 452/D/2010
Tanggal 6 Mei 2010
ADVANCED MULTIFUNCTIONAL INORGANIC
NANOSTRUCTURED OXIDES FOR CONTROLLED
RELEASE AND SENSING
Soumit S. Mandal and Aninda J. Bhattacharyya
Solid State and Structural Chemistry Unit-Indian Institute of Science
Bangalore 560012, India
e-mail: aninda_jb@yahoo.com
ABSTRACT
ADVANCED MULTIFUNCTIONAL INORGANIC NANOSTRUCTURED OXIDES FOR
CONTROLLED RELEASE AND SENSING. We demonstrate here certain examples of multifunctional
nanostructured oxide materials for biotechnological and environmental applications. Various in-house synthesized
homogeneous nanostructured viz. mesoporous and nanotubes silica and titania have been employed for controlled
drug delivery and electrochemical biosensing applications. Confinement of macromolecules such as proteins
studied via electrochemical, thermal and spectroscopic methods showed no detrimental effect on native
protein structure and function, thus suggesting effective utility of oxide nanostructures as bio-encapsulators.
Multi-functionality was demonstrated via employing similar nanostructures for sensing organic water pollutants
e.g. textile dyes.
Key words : Inorganic nanostructured oxides, Nanotubes and mesoporous, Titania, Silica, Controlled in vitro
drug delivery, Biosensing, Water pollutants
ABSTRAK
OKSIDA ANORGANIK BERSTRUKTUR NANO UNGGUL DAN MULTIFUNGSI UNTUK
SISTEM PELEPASAN DAN SENSOR TERKENDALI. Dalam makalah ini didemonstrasikan beberapa
contoh oksida berstruktur nano multifungsi untuk aplikasi di bidang bioteknologi dan lingkungan. Beberapa
material berstruktur nano yang homogen yang telah disintesis termasuk mesoporous dan nanotubes silika serta
titania telah dipergunakan untuk pelepasan obat terkendali dan aplikasi biosensor elektrokimia. Pengenkapsulasian
molekul berukuran makro seperti protein, dipelajari melalui metode elektrokimia, termal dan spektroskopik,
telah menunjukkan tidak adanya efek negatif terhadap struktur asli dan fungsi dari protein, yang menunjukkan
penggunaan yang efektif dari oksida berstruktur nano sebagai bioenkapsulan. Sifat multifungsi ini
didemonstrasikan juga dengan mempergunakan struktur nano yang serupa sebagai sensor untuk polutan organik
yang ada di air, seperti zat pewarna tekstil.
Kata kunci : Oksida anorganik berstruktur nano, Nanotubes dan mesoporous, Titania, Silika, Pengiriman obat
in vitro terkendali, Biosensor, Polutan air
INTRODUCTION
Inorganic nanostructured materials especially
oxides have gained a huge attention in recent times due
to a large set of outstanding properties such as high
surface to volume ratio, relatively easier intrinsic
morphology and chemical composition manipulation,
chemical and physical stability. Major advantage over
organic materials is that many of the beneficial features
described above can be achieved in the same material.
Oxides have been demonstrated as promising materials
for diverse applications ranging from daily life,
biotechnology (targeted drug delivery and biosensing),
optical devices, chemical sensing, inorganic
photovoltaics, photocatalysis, and environmental
detection and cleaning [1-4]. As a result of this versatile
display, R&D laboratories both in the academia and
industry are devoting considerable efforts in synthesis,
characterization and standardization of oxide materials
for specific applications [5]. Inorganic porous materials
with pore size ranging from 2-50 nm have been
demonstrated to serve as excellent hosts/substrates for
various biotechnological and environmental
applications. They are of considerable importance as a
host material for immobilization of a variety of guest
molecules such as proteins, drugs and smaller biological
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Indonesian Journal of Materials Science
molecules (amino acids, peptides, vitamins). It has been
shown that confinement of molecules inside pores of
inorganic nanomaterials does not adversely affect its
function and activity. We discuss here a few examples
of relevance related to biotechnology and environment
(water soluble textile dye). Use of mesoporous oxide
(silica and alumina) materials for in vitro drug and enzyme
inhibitor delivery is highlighted here in considerable
detail. The consequence of protein delivery confinement
inside silica nanotubes (SNTs) and titania nanotube
(TNTs) on protein structure and function (via ligand
binding activity) is also discussed via electrochemical
and thermal stability studies. On the environmental side,
we demonstrate the employment of similar
nanostructured materials (nanorods) for detection and
photocatalytic degradation of commonly used cationic
and anionic textile dyes in aqueous solution.
EXPERIMENTAL METHODS
Nanotubes/nanowires of silica and titania were
synthesized using variety of low temperature methods
(template, chemical solution routes, hydrothermal/
solvothermal). The synthesized nanotubes/nanowires
were homogeneous and morphology i.e. length, pore
size and area were found to be a function of synthesis
methods and conditions. The morphology was
characterized using various electron microscopy
Scanning Electron Microscopy (SEM), Transmission
Electron Microscopy (TEM), nitrogen adsorption/
desorption (BET) and X-Ray Diffraction (XRD).
For the purpose of our study, the SNTs were
synthesised using commercially available anodisc
(alumina) as templates and SiCl4 as the silica precursor.
After repeated cycling the template was dissolved in
strong acid to get the silica nanotubes. These SNTs were
functionalised with various functional groups such as
2,3 Dihydroxynaphthalene (DN) to change its surface
chemical nature. The SNT and SNT-DN were impregnated
with drugs by immersing them in the model drug solution
(IBU) overnight at 4 oC followed by drying at 55 oC
overnight. Similar method but with an appropriate titania
precursor was used for the preparation of TNTs. The
pristine and composite nanotubes were characterised
and used for studying in vitro drug and inhibitor release
kinetics under normal physiological condition as well as
under the influence of an external stimuli such as an
ultrasound impulse. On the other side, these nanotubes
were also incubated for instance with hemoglobin
(abbreviated Hb) protein (human Hb) adequately. For
electrochemical biosensing this solution was drop casted
onto an electrode (glassy carbon, abbreviated as GCE)
followed by drying. Similar procedure was also employed
for pollutant sensing viz.dye sensing. In this case we
had employed TiO2 nanowires synthesised via polyol
technique. Impregnation of the silica nanotubes with
Hb was probed using TEM, UV-Vis and Fourier
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ISSN : 1411-1098
Transform Infrared (FT-IR) spectroscopy. Circular
Dichroism (CD) spectroscopy were utilised to
characterize the state of the protein confined inside
SNTs. Dye detection was carried out using
spectroscopic and electrochemical techniques. The
photochemical reactor used in this study was made of a
Pyrex glass jacketed quartz tube. Ahigh pressure mercury
vapor lamp (HPML) of 125 W (Philips, India) with supply
ballast and capacitor connected in series with the lamp
was placed inside the jacketed quartz tube.
RESULTS AND DISCUSSION
We discuss first the results of the in vitro delivery
of a model drug, ibuprofen (IBU) using SNTs (scheme 1)
and mesoporous matrices of silica and alumina
(scheme 2). SNTs characterized by TEM were found to
be 250 nm in diameter and 10 m in length. The extent of
IBU loading in SNT/SNT-DN was studied using
Thermogravimetry Analysis (TGA), XRD and FT-IR
spectroscopy. Impregnation of the nanotube by drug as
well as Hb was adjudged by comparing the contrast
changes between several transmission electron
micrographs of unloaded and loaded SNTs. The OH
stretch band almost diminishes upon functionalization
with DN, suggesting the successful functionalization of
silanol groups on SNT. Change in the intensity of bands
at 1718 and1417 cm-1 also indicates the interaction of
IBU with SNT. From the TGA analysis, the drug loading
in the bare SNT was estimated to be ~46% and that of
SNT-DN was ~26%. The in-vitro drug release kinetics
from these SNT/SNT-DN IBU composite was carried out
into the simulated body fluid (SBF) (3 mg/mL) at 25 oC
(Figure 1(a)). It was found that release kinetics of IBU
from SNT was ~20% while in case of SNT-DN, after an
initial slow release in the first 13 hours the release goes
up to as high as ~95% in 24 hours showing the effect of
functionalization of SNT with DN. The initial slow release
in case of SNT-DN is attributed to the dominance of
attractive interaction (such as π-π) between the DN
group and IBU over that of solvation of IBU (i.e. the
COOH group) by the aqueous buffer (SBF) and release
increases only when a sufficient amount of solvent
Scheme 1. Silica and titania nanotubes as controlled drug
and enzyme delivery systems
Advanced Multifunctional Inorganic Nanostructured Oxides for Controlled Release and Sensing (Soumit S. Mandal)
(a)
(b)
Figure 1. (a). Kinetics of IBU release in buffer (SBF;
pH = 7.3) from various samples : MCM-48: IBU, SNT :
IBU and SNT-DN:IBU (drug release kinetics observed via
monitoring characteristic peak at 264 nm of IBU by
uv-vis spectroscopy). Inset: TEM images of SNT with
impregnated drug and (b). IBU release in buffer (SBF;
pH = 7.3) from SNT:IBU under ultrasound impulse of 0.5
min duration impulse with 2 min rest time. Inset: Post
kinetics TEM images of SNT.
molecules have diffused through the channels of the
nanotubes for solvating the drug. In order to obtain
higher drug release from pristine SNT, an alternative
would be the employment of external stimuli such
as ultrasound (Figure 1(b)) favoring beneficial
morphological changes. Out of the various ultrasound
impulse protocols, impulses of shorter duration
(~ 0.5 min) and shorter time intervals between
successive impulses were observed to produce higher
drug yields. Since longer pulse duration creates higher
amount of debris from the tubes into which the open
end of the tubes get entrapped or the tube on the whole
may get entrapped which leads to low drug release [5].
Scheme 2. Mesoporous oxide materials for multi-drug
delivery
Figure 2. Release kinetics of IBU from Al2O3-X into buffer
(SBF; pH = 7.0) at 25 oC
The crucial role of the drug carrier surface
chemical moeities on the uptake and in vitro release of
drug are also of considerable importance in case of
mesoporous oxide matrices (Scheme 2). Mesoporous
alumina with a wide pore size distribution (2-7 nm)
functionalized with various hydrophilic and
hydrophobic surface chemical groups was employed as
the carrier for delivery of the model drug ibuprofen.
Surface functionalization (Figure 2) with hydrophobic
groups resulted in low degree of drug loading
(approximately 20 %) and fast rate of release (85 % over
a period of 5 h) whereas hydrophilic groups resulted in
significantly higher drug payloads (21% - 45 %) and
slower rate of release (12 %-40 % over a period of 5 h).
Depending on the chemical moiety, the diffusion
controlled ( time-0.5) drug release was additionally
observed to be dependent on the mode of arrangement
of the functional groups on the alumina surface as well
as on the pore characteristics of the matrix. For all
mesoporous alumina systems the drug dosages were far
lower than the maximum recommended therapeutic
dosages (MRTD) for oral delivery.
The feasibility of utilizing mesoporous matrices
of alumina and silica for the inhibition of enzymatic
activity was also carried out (Figure 3). The studies were
performed on a protein tyrosine phosphatase by the
name chick retinal tyrosine phosphotase-2 (CRYP-2),
a protein that is identical in sequence to the human
glomerular epithelial protein-1 and involved in
hepatic carcinoma. The inhibition of CRYP-2 is of
tremendous therapeutic importance. Inhibition of
catalytic activity was examined using the sustained
delivery of para nitrocatechol sulfate (pNCS) from
bare and amine functionalized mesoporous silica
(MCM-48) and mesoporous alumina (Al 2 O 3 ).
The amine functionalized MCM-48 (Figure 3(a))
exhibited the best release of pNCS and also
inhibition of CRYP-2. The maximum speed of reaction
vmax (= 160 ± 10 µmols/mnt/mg) and inhibition constant
Ki (= 85.0 ± 5.0 µmols) estimated using a competitive
inhibition model were found to be very similar to
inhibition activities of protein tyrosine phosphatases
using other methods (Figures 3(b) and 3(c)).
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(a)
(b)
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ISSN : 1411-1098
(a)
(b)
(c)
Figure 4. (a). Cyclic voltammogram (@ 25 °C; scan rate
= 0.01 V s-1) of SNT, Hb/GCE and SNT-Hb/GCE in 0.1 M
PBS (pH 7.0), (b). Fraction of unfolded Hb (fu) in the
temperature range from (25-85) °C (heating rate = 1°C
min-1) for (a) free Hb in solution (0.1 M PBS, pH 7.0) and
(b) Hb confined inside SNTs
Figure 3. (a). Release kinetics of pNCS (into HEPES
buffer at 25 °C) from Al2O3/Al2O3-NH2/MCM-48/MCM48-NH2 and inhibition, (b). Inhibition kinetics of pNCS
from Al 2 O 3 /Al 2 O 3 -NH 2 /MCM-48/MCM-48-NH 2 and
(c). The effect of pNCS on the CRYP-2 catalyzed pNPP
hydrolysis. The Michealis-Menten plots carried out in
the presence of varying concentrations of pNCS,
0 µmols (
), 50 µmols(
), 100 µmols (
) and
200 µmols (
) indicate competitive inhibition
of CRYP-2 activity by pNCS with a inhibition constant
of 85 ± 5 µmols.
Scheme 3. Silica nanotubes (SNTs) as biosensors and
bio-encapsulators
16
We now discuss the application of SNT and
TNT as substrates for electrochemical biosensors
(Scheme 3). For Hb immobilized inside SNT, the Soret
band is considerably broadened and is slightly red shifted
to 409 nm as against that of free Hb at 406 nm. This shift
of 3 nm is attributed to the interaction of Hb with SNT.
The amide I and amide II bands for SNT-Hb composite
appeared at nearly the same wave numbers as observed
for free Hb in solution. This suggests an unchanged
secondary structure of Hb confined inside the SNTs
which was further confirmed from the CD spectrum which
shows two characteristic bands at 208 and 222 nm that
are characteristic of the -helix structure of Hb [7].
Figure 4(a) shows the cyclic voltammogram (CV)
of Hb/GCE, SNT/GCE, and SNT-Hb/GCE in 0.1 M PBS
buffer solution (pH 7.0). No peaks were observed for
SNT/GCE indicating no electroactivity in the applied
potential range. The Hb/GCE electrode showed the
response of Hb, but only one irreversible redox peak
was observed at -0.40 V. Further, the steady increase in
anodic current suggested some kind of decomposition.
The SNT-Hb/GCE electrode showed a pair of well-defined
reversible peaks at -0.30 and -0.42 V which result from
the electrochemical redox couple: Fe (III)/Fe (II) of Hb.
This strongly suggests that immobilization of Hb inside
SNTs does not result in any distortion of the native
protein structure and activity rather enhances its
electrochemical activity. This fact was further supported
Advanced Multifunctional Inorganic Nanostructured Oxides for Controlled Release and Sensing (Soumit S. Mandal)
by the ligand binding studies using pyridine based model
ligands [7]. Thermal stability studies were carried out by
monitoring the CD signal at 222 nm in the temperature
range (25 - 85) °C. An increase of 4 oC in the denaturation
temperature was observed upon confining Hb inside
SNTs suggesting increased thermal stability of Hb inside
SNTs. We have extended our studies to other class of
proteins viz. cobalt and zinc based proteins. As this is
outside the scope of the paper, we do not discuss theme
any further here.
We now discuss application of titania
nanowires as effective hosts for the detection of
organic water pollutants such as textile dye molecules.
The TiO2 “nanowires” synthesized using polyol method
was found to have an average length of  3 m and
diameter of  800 nm. The XRD pattern was indexed
to the anatase phase of titania. These nanowires
were mesoporous in nature with an average surface area
of 43 m2g-1.
The synthesized titania nanowires (TiNWs)
preferentially observed to adsorb cationic dyes. This
was observed via the adsorbed dye yields (obtained
using uv-vis spectroscopy) on the TiNWs following
dispersing them in aqueous solution containing cationic
methylene blue (MB), anionic OG (anionic) dyes and
their mixtures. The preference for cationic dyes were
confirmed using  -potential measurements which
measured a negative potential for the pristine TiNWs.
Owing to the preferential adsorption of cationic dyes by
(a)
the TiO2 nanowires, we have chosen MB as a model dye
for sensing as well as for photocatalysis studies.
Figure 5(a) shows the electrochemical responses of
various working electrodes: MB/GCE (i.e. GCE dipped in
aqueous MB solution), TiNW/GCE in pure deionised
water and in different concentration of aqueous MB
solutions. The absence of redox peaks in case of TiO2
suggests electroinactivity of the system in the applied
potential window.
The MB/GCE showed very broad (and low
current intensity) reversible peaks at approximately
-0.36 V and -0.29 V. The TiNW /GCE electrode
assembled in aqueous MB solution (50 ppm) also
showed a pair of well defined reversible peaks at -0.31 V
and -0.19 V with enhanced redox peak currents
which were much higher in magnitude compared to
MB/GCE (50 ppm). The electrode reaction of MB
involves two successive one-electron charge transfers
coupled with a rapid reversible protonation between
MB+ and leucomethylene blue (LMB).
The current response was found to vary with
concentration. From the linear plot of cathodic peak
current versus concentration (Figure 5(b)) the sensitivity
of the TiO2/GCE system was found to be 0.003 Appm-1
[8]. The sensitivity was also found to be a function of
NW dimension. The sensitivity can also be further
enhanced via decoration of oxide surface with
metals such as Ag. These nanowires also possessed
excellent photocatalytic abilities compared to the
commercially available P25 (Evonik, formerly Degussa)
titania both under normal and acidic and basic
pH conditions.
CONCLUSIONS
(b)
Figure 5. (a). Cyclic voltammograms at 25 oC (scan rate
of 0.01 V s -1 ) for TiO2 /GCE (in water) MB/GCE and
TiO 2 /GCE (in 50 ppm MB in 100 ml solution) and
(b). Variation of anodic current (from cyclic voltammetry
at 25 oC at scan rate 0.01 Vs-1) with different initial MB
concentrations (0-100 ppm) in 100 ml solution.
We have demonstrated convincingly the
immense importance of inorganic nanostructured
oxides for biotechnological and environmental
applications. The SNTs employed in our studies would
serve as a unique host for the delivery of various
important drugs such as anticancer drugs and
release rates can be altered via optimized morphologies,
surface chemical functionalization and ultrasound
impulses.
The enhancement in the physico-chemical
properties of protein as a result of confinement provides
yet another evidence for the application of these
materials in the fabrication of biosensors and
encapsulators. Employment of TiO2 for environmental
concerns demonstrates yet another application of the
versatile TiO2. Tailoring of surface chemical moieties
(hydroxyl groups of the present study) would further
allow detection of analytes of widely varying sizes and
types. We envisage that the results discussed here will
open up new prospects for inorganic nanomaterials in
diverse applications such as biology, medicine several
other green technologies.
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ACKNOWLEDGMENT
The authors thank I.S. Jarali (SSCU, IISc.,
Bangalore) for TGA, BET and FTIR measurements, Amit
Mondal (INI, IISc., Bangalore ) for TEM and Sumanta
Mukherjee (SSCU, IISc.) for SEM, T.S. Girish and B Gopal
(MBU, IISc) for providing hemoglobin and inhibitor
studies, S. Kapoor, P. Murria and D. Jose (SSCU, IISc.)
for technical discussions. AJB thanks DST, Govt. India
for financial assistance.
Special Edition on Materials for Sensor 2011, page : 13 - 18
ISSN : 1411-1098
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