Colloids and Surfaces A: Physicochem. Eng.

Aspects 455 (2014) 111–121

Contents lists available at ScienceDirect

Colloids and Surfaces A: Physicochemical and

Engineering Aspects
journal homepage: www.elsevier.com/locate/colsurfa

In vitro & in vivo correlation of release behavior of andrographolide

from silica and PEG assisted silica gel matrix
Suparna Chakraborty a,∗ , Supratim Biswas b , Biswanath Sa c , Satadal Das d , Rajib Dey b
a
School of Materials Science and Nanotechnology, Jadavpur University, Kolkata 700032, India
b
Department of Metallurgical and Material Engineering, Jadavpur University, Kolkata 700032, India
c
Department of Pharmaceutical Technology, Jadavpur University, Kolkata 700032, India
d
Department of Microbiology, Peerless Hospital & B. K. Roy Research Centre, Kolkata 700094, India

h i g h l i g h t s g r a p h i c a l a b s t r a c t

• Higher PEG percentage decreases

surface area while increases average
pore diameter.
• The in vitro drug release was found to
be biphasic (initial burst followed by
slow release).
• Increasing percentage of PEG facili-
tates higher release and availability of
the molecule in blood.
• The drug release profile establishes
Level A in vitro–in vivo correlation.
• No degenerative histological changes
associated with silica carriers are
found.

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

Article history: Silica xerogel and its PEG assisted derivatives were prepared by sol–gel method for controlled release of
Received 17 December 2013 andrographolide. In vitro and in vivo release of andrographolide from the nano porous silica as well as PEG
Received in revised form 14 April 2014 modified silica matrix were studied. Drug release from the matrix increased with increasing percentage
Accepted 18 April 2014
of PEG and followed a biphasic pattern. The in vitro release profile followed zero order kinetics with
Available online 26 April 2014
erosion of the matrix for the first six hours and higuchi model with the diffusion mechanism for rest of
the time period. Pharmacokinetic data revealed sustained release of the drug from silica–drug composite
Keywords:
with higher elimination t1/2 than the pure drug. For all the formulations Level A in vitro-in vivo correlation
Nano porous silica gel
Andrographolide
(IVIVC) was established with (R2 ) > 0.98. Histology of different organs was carried out following in vivo
PEG administration of the drug-carrier composite. The histological examination demonstrated absence of
Sustained release any inflammatory or degenerative response with the formulations. Fourier transform infrared (FTIR)
IVIVC data revealed the coexistence of andrographolide in the silica matrix. Transmission electron microscope
(TEM) images also unfold the repose matrix structure in PEG assisted derivatives than the pure matrix.
This study discloses that silica gel and PEG assisted derivatives could be used as a biocompatible and
sustained release device for andrographolide.
© 2014 Elsevier B.V. All rights reserved.

1. Introduction

∗ Corresponding author. Tel.: +91 9477270256. Sustained release emphasizes on delivering therapeutic
E-mail address: riyachak329@gmail.com (S. Chakraborty). molecule or biomolecules over an extended period of time.
http://dx.doi.org/10.1016/j.colsurfa.2014.04.046
0927-7757/© 2014 Elsevier B.V. All rights reserved.
112 S. Chakraborty et al. / Colloids and Surfaces A: Physicochem. Eng. Aspects 455 (2014) 111–121
Prerequisite for delivery of molecules in a sustained manner are
resorption of drug carrier material and diffusion. To suppress the
systemic toxicity and damage of tissues due to carrier molecule
leads to the immediate endeavor for an ideally biodegradable mate-
rial possessing the property of biocompatibility [1]. Amorphous
nanoporous silica exhibits unique physicochemical properties and
due to its room-temperature synthesis, it becomes favorable for the
tissue response, making it a potential drug carrier in the biomedical
field [1]. Silica encapsulation contributes to longer residence time
in circulation, protection from degradation and providing greater
therapeutic efficiency of drug [2]. Other unique features of the
system include non-interfering drug activity, capacity of delivering
a therapeutic dose of drug, homogenous drug distribution into the
system [2,7]. Synthesis of silica gel through sol–gel route involves
a series of reactions, which includes hydrolysis and condensation
[3–5]. Porous silica offers a major prevalence over non-porous
high surface area materials, where the deposited molecules are
not only adsorbed onto the silica surface but also confines into
the pores [6]. Drug release by the virtue of matrix degradation
to a higher extent would have been necessary if the doped
molecule is larger than the average pore size or entrapped within
an isolated pore. Organic additives are used during the sol–gel
process as a technique to control the pore size of silica gel [6].
Polyethylene glycol (PEG) is one of the most well known polymers
(organic additive) used in chemical and medical sciences due to
its hydrophilicity, flexibility, lack of toxicity, non-immunogenicity
and easy availability [6,8,9]. PEG polymers have ether oxygen
atoms (hydrogen-bonding acceptor) and a high affinity toward
silanol groups (hydrogen-bonding donor) of silica species through
hydrogen-bonding interactions in the solution at almost neutral
pH value and ambient temperature [10,11]. PEG polymer finds
widespread use as flocculants and structure directing agents
in preparation of silica gel and microporous silica sphere [11].
Properties like hydrogen bonding interactions between PEG and
silica, [12] the mobility of PEG upon immersion of PEG-templated
silica in water solution can be manipulated by temperature or
pH [10]. Under physiological conditions facilitating drug release,
PEG-templated silica can provide an avenue for formation of larger
pores in these materials by breakage of the hydrogen bonds [6].
As PEG is a water-soluble agent, it can be liberated from materials
spontaneously resulting in complete release of a drug from the
sol–gel matrix, perhaps without a higher burst release of the drug
[8]. In this present study, low molecular organic polymer, PEG
400 (where 400 indicates the average molecular weight) was
used as additive to prepare the silica gel through sol gel route
for modifying the matrix structure [8,11]. Kinetic models were
employed to evaluate the drug dissolution and modified release
dosage forms along with their in vitro release mechanism [12].
Both the in vitro dissolution characteristics of the drug and its
in vivo bioavailability are the factors which govern the therapeutic
efficacy of pharmaceutical formulations [13].
In vitro–in vivo correlation (IVIVC) is associated with the rela-
tionships between the in vitro dissolution of a dosage form and
the in vivo input rate of a drug released from the same dosage
form. IVIVC is regularly used as both a method for evaluating prod-
uct quality following device preparation and as a surrogate for
predicting the biological performance of a dosage form in vivo.
They are particularly valuable since they can minimize the num-
ber of in vivo (human and animal) studies required for dosage form
development [13,14]. The United States pharmacopeia (USP) estab-
lished different levels of correlation, designated as A, B and C, in
decreasing order of preferences and acceptability, which depends
on the method used to correlate the data. Level A IVIVC represents
a point-to-point correlation between the in vivo absorption pro-
file and the in vitro release profile. Convolution or deconvolution
methods were employed to calculate the in vivo absorption profile
from plasma concentration–time curves. Attempts should be made
to correlate at level B in case level A correlation cannot be estab-
lished. Level B correlations are based on statistical moment analysis
and compare the mean in vitro dissolution time of the formulation
with either the mean residence time in the body or the mean in vivo
dissolution time of the formulation. Level B IVIVC is known to be
useful for extended release products, although a single parameter
is compared in this method. Level C IVIVC yields less information
which may find usefulness in formulation development. This type
of correlation represents a single-point relationship and compares,
e.g. the time required for 50% drug release in vitro with the area
under the concentration time curve [14,15].
Andrographolide is a diterpene lactone, colorless, crystalline,
and bitter in taste and it is known for anti-parasitic, anti-bacterial,
anti-retroviral, anti-diabetic, pro-apoptotic, etc. properties. It is
sparingly soluble in water due to which it’s bio-distribution and
localization is limited. The plasma half life (t1/2 ) is also very short
[16]. These are the reason behind encapsulation of the same into the
silica gel matrix. Objectives of the present study were to evaluate (i)
the capability of silica gel to deliver andrographolide in a sustained
manner, (ii) in vitro and in vivo kinetics of release, their mechanism
and correlation and (iii) the tissue distribution of andrographolide.
2. Experimental
Tetraethyl orthosilicate (TEOS, reagent grade, 98%) and andro-
grapholide (98%) were purchased from Sigma–Aldrich and used
without further purification. Absolute ethanol procured from
Merck Germany and polyethylene glycol was purchased from
Merck specialties private limited (Mumbai) and used as received.
Sodium chloride (NaCl), sodium hydrogen carbonate (NaHCO3 ),
sodium sulfate (Na2 SO4 ), potassium chloride (KCl), di-potassium
hydrogen phosphate anhydrous (K2 HPO4 ), magnesium chloride
hexahydrate (MgCl2 ·6H2 O), calcium chloride (CaCl2 ) and tris-
buffer were purchased from Merck specialties private limited
(Mumbai) for SBF preparation. Deionized water (reagent grade)
from hydro lab, Ruby Park, Kolkata was used throughout the exper-
imental procedure.
2.1. Preparation of sol–gel processed silica gel particles
2.1.1. Silica gel with andrographolide
Silica gel particles were prepared at room temperature by
hydrolysis and polycondensation of tetraethylorthosilicate (TEOS),
water and ethanol in a mole ratio of TEOS: H2 O:C2 H5 OH = 1:4:2.
This ratio, following a series of preformulation studies, was found
optimum. The weight ratio of andrographolide to silica was 7%. The
TEOS and ethanol solution was magnetically stirred at 300 rpm for
30 min followed by the addition of 2 mg andrographolide, water
and ethanol and stirring was continued at the same speed until
a clear solution was obtained. There after it was covered with a
parafilm and left for gelation [17–19]. Andrographolide and silica
gel entrapped andrographolide were referred A and P0 in the rest
of the article.
2.1.2. Silica/PEG with andrographolide
Low molecular weight polyethylene glycol (PEG) (400 g/mol)
was taken due to better water solubility, and it is easier to excrete
out from the body. As for polymers with higher molecular weight
accumulation in the liver takes place leading to macromolecular
syndrome. Gels were prepared by co-hydrolysis of TEOS, PEG with
water and ethanol in a mole ratio of 1:a:4:2, where ‘a’ is the mole
ratio of PEG whose values are 0.03, 0.06, 0.09, and 0.12. Formu-
lations were indicated as P1, P2, P3 and P4 indicating increasing
 S. Chakraborty et al. / Colloids and Surfaces A: Physicochem. Eng. Aspects 455 (2014) 111–121
percentage of PEG [8,17–19]. In each formulation, the weight ratio
of andrographolide to silica was 7%.
2.2. Characterizations of samples
Different instrumental facilities used during the experimental
procedures described in the present manuscript are mentioned
here under.
In vitro drug dissolution was done by using USP II dissolution test
apparatus model TDP-06P, Electrolab, Mumbai; UV–visible spec-
troscopic analysis was done using Perkin Elmer model Lambda 35
instrument; Infra-red spectroscopic analysis was done using Schi-
madzu model prestige22 instrument. The specific surface areas
were determined using the Brunauer–Emmet–Teller (BET) method
by using Gemini VII 2390 t surface area analyzer. TEM analysis was
done by using HRTEM JEOL-TEM 2100. XRD analysis was done using
a Rigaku Ultima III. Light microscopy was done by LEITZ PERIPLAN
GF, model no 081465.
2.3. In vitro dissolution test
Silica gel particles were immersed in 500 mL of SBF buffer of pH
7.4, prepared to have an ion concentration nearly equal to that of
human blood plasma (Na+ – 142.0, K+ – 5.0, Ca 2+ – 2.5, Mg2+ – 1.5,
Cl− – 147.8, HCO3 − – 4.2, HPO4 2− – 1.0, and SO4 2− – 0.5 mM) fol-
lowing the method described by Kokubo et al., 2003 at 37 ◦ C under
rotation at a rate of 75 rpm. Aliquots (3 mL) of sample were removed
for analysis at given time intervals and replaced with the same
volume of fresh SBF (simulated body fluid) solution. The total vol-
ume of SBF-buffer for each sample was regularly replaced with an
equal volume of pre-thermostatted SBF, every 24 h during release.
The behavior of drug release was observed for 168 h [20–22]. The
collected solution was analyzed by UV–Vis spectroscope at a wave-
length of 227 nm. This method obeyed Beer’s law and the amount of
andrographolide released was determined using a calibration curve
which was prepared using different amounts of andrographolide in
the range of 1–20 ␮g [20–22].
The results obtained from in vitro release studies were plotted in
different models. According to the zero-order equation (cumulative
amount of drug release versus time) the drug release rate is inde-
pendent of its concentration. The equation is C = K0 t, where K0 is the
zero-order rate constant expressed in units of concentration/time
and t is the time.
Release rate is concentration dependent according to the first-
order equation (log cumulative percentage of drug remaining to
be released versus time). The mathematical expression of the first
order equation is log C = log C0 − kt/2.303, where C0 is the initial
concentration of drug and K is first order constant.
Higuchi model (cumulative percentage of release versus square
root of time) describes the release of drugs from the insoluble
matrix as a square root of time dependent process based on Fick-
ian diffusion and the equation is Q = Kt1/2 , where K is the constant
reflecting the design variables of the system. Korsmeyer–Peppas
equation (log cumulative percentage of drug released versus log
time) derived a simple relationship which described drug release
from a polymeric system.
From the equation Mt /M∞ = Ktn , “n” values can be used to charac-
terize the mechanism of drug release. The value of “n” 0.45 indicates
Fickian diffusion; 0.45 < n < 0.89, anomalous (non-Fickian) diffu-
sion; 0.89, case II transport; and n > 0.89, super case II transport
[22,23].
2.4. In vivo release study
Fifteen (2 months old) male Sprague Dawley albino rats were
procured from Indian Institute of Chemical Biology, Council of
113
Scientific & Industrial Research (CSIR) laboratory, Kolkata for the
present study. The animals were kept in the animal house for 7 days
prior to the initiation of the study. All animals were clinically exam-
ined upon arrival and any animal showing signs of abnormality or
disease was excluded.
A total number of 15 male rats were divided into three groups
containing five in each. They were injected intraperitoneally with
silica and PEG assisted silica loaded with andrographolide at a con-
centration of 2 mg/kg body weight while only andrographolide was
injected at a concentration of 20 mg/kg body weight. All injectable
were suspended in 0.5 mL physiological saline prior to adminis-
tration. Blood samples drawn from the tail vein were collected
at a predetermined time interval in EDTA-coated tubes and cen-
trifuged; the prepared plasma was stored at −20 ◦ C prior to analysis
using an UV–visible spectrophotometer. Animals were euthanized
on 8th day of administration of the initial dose. Heart, lung, spleen,
liver, kidney and intestine were collected for histological analysis.
Serum concentrations were used to determine various pharmacoki-
netic parameters. The area under the plasma concentration–time
curve from zero to the last measurable plasma concentration at
time ‘t’ (AUC0−t ) was calculated using the linear trapezoidal rule.
The area was extrapolated to infinity (AUC0−∞ ) by addition of Ct/kel
to AUC0−t , where Ct is the last detectable drug concentration. The
first order elimination rate constant (kel ) was estimated by the least
square regression of plasma concentration vs. time data points of
the curves describing the terminal log-linear decaying phase. Elim-
ination half life (t1/2 ) was derived from kel (t1/2 = ln 2/kel , where ln is
the natural logarithm). The absorption rate constant (ka ) was deter-
mined by the residual method. The AUMC is the area under the plot
of time vs. the product of time and concentration extrapolated to
infinity and was calculated by trapezoidal rule. The mean residence
time (MRT) was determined by AUMC divided by AUC. The maxi-
mum observed andrographolide concentration (Cmax ) and the time
at which Cmax was observed (Tmax ) were reported directly from the
profile. The fraction absorbed (fabs ) for each formulation was then
calculated by using the mean (n = 5) plasma concentration vs. time
profile for each formulation. Finally, the fraction of andrographolide
absorbed (fabs ) in vivo was plotted against the fraction released (frel )
in vitro at corresponding time points. The slope, intercept, and cor-
relation coefficient describing the relationship between the mean
fabs and frel were then determined by linear regression [14,15,24].
2.5. In vitro–in vivo correlation (IVIVC)
For level A IVIVC, the fraction absorbed in vivo was plotted versus
the fraction released in vitro at the same time. Correlation A rep-
resents a point-to-point relationship between in vitro dissolution
and the in vivo absorption. In vivo absorption profiles from plasma
concentration were calculated and correlated with the percentage
released in vitro using a basic linear model with intercept (a) and
slope (b) [13–15].
(% absorbed)in vivo = a + b (% released)In vitro
if (b) = (1); indicates (1:1 correlation)
(b) = (−); indicates (in vivo process lags behind in vitro dissolution)
(b) = (+); indicates (has no clear meaning).
2.6. Histological effects of injected silica gel
The Heart, lung, spleen, liver, kidney and intestine were
removed and fixed in 10% formol-saline, embedded in paraffin,
sectioned and stained with hematoxylin and eosin for histological
examination using standard techniques. Samples were evaluated
using a light microscope.
114 S. Chakraborty et al. / Colloids and Surfaces A: Physicochem. Eng. Aspects 455 (2014) 111–121
3. Results and discussions
3.1. TEM analysis
Fig. 1 shows the morphology of silica gel entrapped andro-
grapholide (A) and PEG assisted silica gel-entrapped andro-
grapholide (B). PEG is an organic polymer known to have the
ability to decrease the dielectric constant of the solvent thus in
turn induces particle aggregation leading to relaxation of inter-
nal tension in the silica network which might have resulted in the
formation of branched structure with larger mesopores [8]. TEM
micrograph suggested crystalline patches found within the amor-
phous matrix of silica was andrographolide as shown in Fig. 2C
which was reconfirmed from the d-values obtained from XRD anal-
ysis [25].
3.2. Infra-red analysis
The Fourier transform infrared (FTIR) spectra of silica gel, and
gel-entrapped andrographolide were taken using an FTIR spec-
troscope. Fig. 2 represents the FTIR spectra of bare silica gel,
PEG assisted silica gel, PEG assisted silica gel loaded with andro-
grapholide, within the range of 4000–400 cm−1 (A). In the three
samples; the peaks at 468 and 798 cm−1 were found for the Si O Si
bending vibration; the peak at 950 cm−1 and at 1078 cm−1 were
due to the Si OH stretching vibrations and Si OR stretching vibra-
tion, respectively [26,27]. For pure PEG, (B) the band appearing
at 3555 cm−1 was due to the presence of bonded hydroxyl group
Fig. 1. TEM images of silica-andrographolide matrix (A), PEG assisted silica-andrographolide matrix (B), crystalline andrographolide entrapped into the pore of silica gel (C)
and XRD graph of andrographolide (C inset).
vibrations of the polymer, at 2889 cm−1 was due to CH2 sym-
metrical stretching vibrations. A strong bond for conjugated C C
was appeared at 1650 cm−1 , weak bands at 1356 cm−1 due to
in plane scissoring of the CH2 group, 1256 cm−1 arose for O H
deflection and strong peak at 1151 cm−1 of C O stretch of ether
coming from PEG. In PEG modified bare silica gel, in addition to
the peaks of silica three distinct peaks 1647 cm−1 , 2934 cm−1 and
3402 cm−1 were obtained, which corresponds to the presence of
PEG [8,28,29]. In pure andrographolide (C) characteristic vibra-
tional peak at 3325 cm−1 due to a hydroxyl group, stretching mode
appearing at 1710 for C O, peak at 1688 cm−1 was due to lactone
ring, peak at 1446 cm−1 appeared due to C C, peak at 1254 cm−1
due to C O, peak at 1021 cm−1 was due to presence of exocyclic
methylene group, peak at 713 cm−1 was due to C C C and band at
568 cm−1 was found to the in-plane and out-of-plane deformations,
respectively, of the carbonyl group were observed [29,30]. The
peaks at 3392 cm−1 , 2926 cm−1 and 1668 cm−1 revealed the pres-
ence of andrographolide on to the PEG modified silica matrix. The
infrared spectra of the PEG-modified andrographolide entrapped
gel showed no new bonds, indicating that the linkage between PEG
and silica sol particles was by molecular forces or hydrogen bond.
Incorporation of PEG in the silica matrix acquired more hydrophilic-
ity due to formation of hydrogen bond [31,32].
3.3. Surface area analysis
The surface areas were given in Table 1. PEG percentage is
inversely proportional to surface area and pore volume. Pore
 S. Chakraborty et al. / Colloids and Surfaces A: Physicochem. Eng. Aspects 455 (2014) 111–121 115

Fig. 2. FTIR spectrum of bare silica, PEG-silica, PEG assisted silica entrapped andrographolide (A), FTIR spectrum of PEG (B), FTIR spectrum of andrographolide (C).

volume decreased from 0.92 to 0.34 cm3 /g and an increase in aver- due to the entrapment or bounding of andrographolide in the
age pore diameter greater than 2 nm to almost 10 nm in case of all porous silica matrix [26,30,33]. Within the inner part of the gel,
formulations (P0, P1, P2, P3, and P4) respectively were observed the three-dimensional network of silica effectively immobilized
[8,32]. andrographolide molecules. The steric constrain of the matrix made
difficult for the medicine to elute completely from the matrix to the
surrounding fluid [8,30,33]. Since the typical equilibrium solubility
3.4. Release study and kinetics
at physiological pH (pH = 7.4) of amorphous silica is approximately
120 ppm [8], resulting in rapid achievement of the saturation level
3.4.1. In vitro dissolution kinetic analysis of silica carriers
of silica was found under the desired conditions of drug release. This
The in vitro release experiments were carried out in simu-
was the reason for incomplete drug release, especially the fraction
lated body fluid (SBF). The amounts of andrographolide released
that is entrapped in the isolated pores. The in vitro drug release
in SBF buffer from the silica gel matrix were measured at 227 nm
profiles of the andrographolide from the gel and that from the PEG
(Fig. 3A) (max of andrographolide at 227 nm), then plotted against
assisted silica gel were distinctly different at pH 7.4. Incorporation
time, and the concentrations were calculated from the standard
of PEG in the silica matrix allows the formation of a more intercon-
curves. The release kinetics in both the cases (PEG assisted and
nected porous network. Thus, the solution freely penetrates into
unassisted) was biphasic [22,32] initially it was very rapid in first
the pores of matrices due to the presence of broad channel, result-
6 h while a sustained release was observed at the second phase
ing in reduced internal tension in the silica network. Subsequently,
and gradually slowed down at around 168 h (Fig. 3). The first
andrographolide leached out without any obstacles [8,10,33]. Fig. 3
phase of burst release was due to the release of andrographolide
shows cumulative release of andrographolide from the silica matrix
which was adsorbed superficially on the outer surface, while the
as a function of time. The highest burst effect was observed for the
slowing down of release in the second release phase might be
sample P4, and close to 30.2% of the drug dose was released during
the first 6 h. The initial releases of andrographolide in first 6 h from
Table 1 rest of the formulations were 25.8%, 22.2%, 17.6% and 16% for P3,
Surface area of silica and PEG-silica formulations. P2, P1, and P0 respectively (Fig. 3B). The total percentage of andro-
Sample name PEG (%) Surface area Pore volume Average pore grapholide released during 168 h in SBF were 54.46%, 63.8%, 70.81%
(m2 /g) (cm3 /g) diameter (nm) and 80.2% for P1, P2, P3 and P4, respectively (Fig. 3). The release
P0 – 900 0.92 2.556
percentage of andrographolide in P0 was 46% during the experi-
P1 3 540.82 0.76 5.23 mental time period (168 h). The rate of release of andrographolide
P2 6 460.77 0.58 7.19 increased with an increase in the amount of PEG. The porosity of
P3 9 400.93 0.47 8.52 the drug incorporated substrate also played an important role in
P4 12 339.74 0.34 10.12
the release kinetics. Release of the entrapped agent from the silica
116 S. Chakraborty et al. / Colloids and Surfaces A: Physicochem. Eng. Aspects 455 (2014) 111–121

Fig. 3. In vitro dissolution profile of andrographolide from P0, P1, P2, P3, P4 formulations upto 168 h. Inset A shows the absorbance maxima of andrographolide at 227 nm.
Inset B shows the magnified In vitro dissolution profile of andrographolide from P0, P1, P2, P3, P4 formulations upto 6 h. The data are the five repeat experiments of each set.
Zero order release model of andrographolide in first 6 h (C) and Higuchi release mode of andrographolide in 12–168 h from the formulations (D).

matrix has been found to occur through diffusion-cum-degradation to determine the kinetics of andrographolide release from silica
mediated process [23]. Highly branched network structures of poly- matrix and PEG assisted silica matrix, the dissolution profiles were
meric materials derived from sol–gel method are advantageous as fitted in different mathematical models. The in vitro data (values
they provide gradual, time-dependent release [8,22,30,33]. In order obtained from 1 h to 168 h drug release) of the formulations were

Table 2
Comparative characteristics of regression values of different release kinetic models obtained for andrographolide eluted from the formulations.

Formulations Zero order regression coefficient R2 First order regression coefficient R2 Higuchi’s plot regression coefficient R2 Koresmeyer plot

Regression coefficient n value

Upto 6 h
P0 0.993 0.977 0.983 0.991 0.95
P1 0.991 0.983 0.98 0.99 0.94
P2 0.994 0.976 0.98 0.993 0.95
P3 0.992 0.986 0.986 0.99 0.98
P4 0.996 0.98 0.976 0.991 0.97

After 6 h
P0 0.978 0.98 0.997 0.99 0.286
P1 0.97 0.987 0.997 0.98 0.28
P2 0.976 0.983 0.993 0.98 0.28
P3 0.975 0.986 0.996 0.985 0.284
P4 0.978 0.985 0.995 0.988 0.28
 S. Chakraborty et al. / Colloids and Surfaces A: Physicochem. Eng. Aspects 455 (2014) 111–121 117

Fig. 4. Comparative in vivo profiles of andrographolide. Mean (n = 5) plasma concentration of eluted andrographolide from the formulations vs. time profile after administration
of single dose in white albino rats. Inset A shows mean (n = 5) plasma concentration of andrographolide vs. time profile after administration of single dose in white albino
rats. Relationship between fraction dissolved in vitro vs. fraction absorbed in vivo for P0, P1, P2, P3 and P4 derived from pharmacokinetic profile.

fitted to zero-order, first-order, Higuchi, and Korsmeyer–Peppas
models [22,23]. The model that best fitted with the release data was
evaluated based on the correlation coefficient (R2 ); the R2 values
obtained for all formulations in various models are given in Table 2.
During the initial period (for first 6 h) all the formulations (P0–P4)
followed zero order release kinetics (Fig. 3C) with regression values
(R2 ) >0.99. During the later period (12–168 h) all the formulations
(P0–P4) showed fair linearity in Higuchi’s model (Fig. 3D), with
regression values (R2 ) 0.99. To establish this biphasic kinetics of
drug release; we fitted the data in Korsmeyer–Peppas equation for
the samples (P0–P4). It was found that during the burst period (for
the first 6 h), the release followed the super case II transport, which
was evident from the “n” values (≥0.9), while in a second phase
(12–168 h), the n value was <0.45, suggesting that a Fickian diffu-
sion release was predominant and the drug release occurred by a
diffusion and erosion mechanism [22]. Interestingly, not only the
slope, but also the shape of the release curves was modified, indicat-
ing refinements in the underlying kinetic phenomenon despite of
the identical composition of the organic–inorganic hybrid matrix.
The observed variances could be explained on the fact that the sur-
face interactions of the drug as well as the diffusion barriers of the
matrix control the release kinetics of the drug molecules [30,34].
3.4.2. In vivo pharmacokinetic evaluation of silica carriers
The concentrations of drug in plasma were monitored for 168 h
after the intraperitoneally administrated pure andrographolide,
andrographolide silica and their PEG assisted derivatives. The mean
plasma concentration profiles are provided in Fig. 4 and the mean
pharmacokinetic parameters for the drug and drug entrapped
matrix are summarized in Table 3. The results were analyzed using a
non-compartmental statistical design. These data were of consider-
able interest as it demonstrates the controlled release under in vivo
conditions. The prolonged and out stretched serum profile (Fig. 4)
observed in this study should minimize the high drug concentra-
tions which are associated with adverse secondary complications.
This type of pharmacokinetic profile is usual for sustained release
preparations and indicates that andrographolide would stay within
the therapeutic territory for an extended period of time [14].
118 S. Chakraborty et al. / Colloids and Surfaces A: Physicochem. Eng. Aspects 455 (2014) 111–121

Table 3
Pharmacokinetic parameters for andrographolide after intraperitoneal administration in albino rats (n = 5) of A and P0, P1, P2, P3 and P4 formulations.

Formulations AUC0−∞ (ng h/ml) AUMC0−∞ (ng h/ml) Cmax (ng/ml) Tmax (h) MRT (h) ka (h−1 ) kel (h−1 ) Elimination t1/2 (h)

A 2318.65 10,917 417 4 4.7 1.3 0.23 3.15

P0 35,419.08 970,837 94.6 24 27.41 0.11 0.037 19
P1 39,368.8783.44 1,306,653 115 24 33.19 0.14 0.031 23
P2 41331.4 1,669,792 135 24 40.4 0.21 0.024 28
P3 45,534 2,496,630 166 24 54.83 0.25 0.018 38
P4 48,773 3,267,791 176 24 67 0.32 0.015 46.2

As shown in Table 3, Cmax , ka , and kel of all the formulations (P0, bioequivalence. Level A IVIVC was investigated using linear
P1, P2, P3 and P4) were significantly lower whereas MRT and Tmax regression and the results shown in Fig. 4 (P0–P4). Significant point-
were significantly higher than the crude material (A) though the to-point relationship was observed with regression coefficient
applied dose of crude material was 10 times higher than that of the (R2 >) of 0.98 and slope approaching toward unity, indicating a close
formulations [14]. An essential feature of the encasement is that it correlation between the in vitro release rates and in vivo absorp-
facilitates prolonged availability of the active drug in the blood cir- tion of the drug [40]. The level A IVIVC established in this study
culation which helps in cultivating the therapeutic concentrations confirmed the efficiency of proposed in vitro model in simulating
of the active drug for extended periods, consecutively reducing in vivo condition. Since it represents a point-to-point relationship
dosages and frequency of administration [35]. The elimination t1/2 between in vitro dissolution and the in vivo input rate, this kind of
value of the pure drug was found to be 3.15 h. Incorporation of correlation is quite important for the drug from the dosage form
the drug in silica nanoporous gel increased the elimination t1/2 by [13–15].
about 6-fold. Consequently, while Kel , Ka and Cmax decreased by
6.21-fold, 11.8-fold and 4.4-fold, respectively, AUC0-∞ , Tmax and 3.5. Histological changes in different organs
MRT increased by 15.27-fold, 6-fold, 5.83-fold, respectively. This
indicates the effectiveness of silica–drug formulation as a sustained Histological observations are summarized in Table 4 and rep-
release drug delivery system. Metal oxides have a hydroxyl group resentative micrograph are given in Fig. 5. Histology of kidney
in their surfaces rendering them with hydrophilic property. This showed profound changes, which include glomerulitis, necrosis,
natural hydrophilicity decreased oxide particle clearance resulting degeneration and hyperaemia for andrographolide whereas neg-
increased residence time in blood which intern elevated “elimina- ligible changes were found in silica and PEG assisted silica gel
tion t1/2 ” of silica–drug composite [36]. However, incorporation of
PEG in silica drug formulation increased MRT {P1 (7.06-fold) < P2 Table 4
(8.65-fold) < P3 (11.66-fold) < P4 (14.25-fold)} and AUC {P1 (16.97- Histological changes in different organs after administration of andrographolide,
fold) < P2 (17.82-fold) < P3 (19.63-fold) < P4 (21.03-fold)} values silica gel entrapped Andrographolide, PEG assisted silica gel entrapped Andro-
grapholide. [Note: – no pathological changes, 1+, 2+, 3+, 4+ different degrees of
while decreased Cmax {P1 (3.62-fold) > P2 (3.08-fold) > P3 (2.5-
changes. A = andrographolide, SA = silica gel entrapped andrographolide, S = silica
fold) > P4 (2.37-fold)}. The aspect which has evolved in case of gel, SAP = PEG assisted silica gel entrapped andrographolide].
PEG assisted silica gel from the data set of Table 3 reveals that
Different organ tissues A SA SAP
with increasing percentage of PEG in the formulation leads to the
simultaneous increase in AUC, MRT indicating a prolonged uninter- Kidney
rupted release of andrographolide from the silica matrix making Glomerulitis 3+ 2+ 1+
Necrosis of glomerular cell 2+ – –
it a sustained release system. Another interesting fact observed Necrosis of bowman’s capsule 2+ – –
was the decreasing trend of Kel from A to (P1–P4) by 7.42, 9.58, Degeneration of convoluted tubules 4+ 1+ 1+
12.77 and 15.34-folds respectively. On the other hand an increas- Necrosis of tubular cells 2+ – –
ing trend of elimination t1/2 was observed with increasing amount Hyperaemia of vessels 3+ 2+ 1+
Black particles – 2+ 1+
of PEG by 7.3, 8.8, 12, 14.7-folds for P1, P2, P3, and P4 respectively
indicating longer residence of andrographolide in the circulation Liver
establishing sustained delivery property of the system. The pro- Degenerative changes 3+ 2+ 1+
Apoptosis 3+ – –
longed circulation time that PEG bestows upon andrographolide is
Necrosis 1+ – –
through decrease in the rate of kidney clearance and an increase Intercellular space enlargement 3+ 1+ 1+
in protection from enzymatic degradation, both of which decrease Black particles – 2+ 1+
the overall clearance of the entrapped molecule. Presence of PEG Spleen
in the silica gel increases hydrophilicity at neutral pH. In contact Red pulp changes 3+ 2+ 1+
with aqueous fluid, PEG dissolves quickly leading to matrix degra- White pulp changes 3+ 2+ 1+
dation resulting increased percentage release of andrographolide Black particles – 2+ 1+
in comparison to pure silica matrix [37–39]. Biocompatibility and Lung
clearance of silica from circulation are provided in Supplementary Alveolar exudate 2+ 1+ 1+
documents S1. Interalveolar space exudate 2+ 1+ 1+
Hyperaemia of blood vessels 2+ 1+ 1+
Black particles – 3+ 2+
3.4.3. Establishment of in vitro/in vivo correlation
Heart
During pharmaceutical development correlations between
Degenerative changes 2+ – –
in vitro and in vivo data (IVIVC) are often used in order to opti- Necrotic changes 2+ – –
mize the formulation while reducing product development time Hyperaemia of blood vessels 3+ – –
and costs. A well correlation is a tool for predicting in vivo results Black particles – 2+ 1+
based on in vitro data and it allows dosage form optimization with Intestine
the fewest possible trials [13–15]. Mucosal inflammation 2+ – –
Establishment of an IVIVC can add in vivo meaning to the Ulceration 2+ – –
Black particles – 3+ 2+
in vitro dissolution test and can be useful as a surrogate for
 S. Chakraborty et al. / Colloids and Surfaces A: Physicochem. Eng. Aspects 455 (2014) 111–121 119

Fig. 5. Representative microscopic images of histological section of tissues from different organs subjected to andrographolide, silica andrographolide and PEG assisted silica
andrographolide.

entrapped andrographolide treated animals. Necrosis was absent
in silica and PEG assisted silica derivatives. Degenerative changes
and intercellular space enlargement along with apoptosis were
markedly present in liver cells of andrographolide treated rats,
while negligible changes were observed in liver cells of rats treated
with silica, and its PEG assisted derivative. Nonspecific reactive
changes in red and white pulp were profound in andrographolide
treated spleen cells, which was found to be moderate in case of sil-
ica gel while some minor changes were found with PEG assisted
silica gel. Inflammatory alveolar exudates, interalveolar space exu-
dates and hyperaemia of blood vessels were moderately present
in andrographolide treated lung cells while mild changes were
found in silica and PEG assisted form. Inflammatory degenerative,
necrotic changes and hyperaemia of blood vessels were evident in
andrographolide treated heart cells whereas such changes were not
found in silica gel and in PEG assisted form. Mucosal inflammation
and ulceration in intestinal cells were observed in animals treated
with crude drug where the same were not observed in silica and
120 S. Chakraborty et al. / Colloids and Surfaces A: Physicochem. Eng. Aspects 455 (2014) 111–121
PEG assisted formulations. Histological changes were more due to
high dose of crude material while the changes were less with silica
entrapped molecule. Release profiles suggested that more erosion
of silica gel occurs due to hydrophilicity of PEG polymer leading
to higher release of entrapped molecules from the matrix. Least
changes or sometimes no changes in histology associated with PEG
assisted silica carrier in comparison to without PEG formulation
were found due to its instauration property exhibited in blood pH
(7.4). Deposition of silica particles in various organs observed by the
virtue of the presence of black particles clearly indicates formula-
tions without PEG exhibiting lower instauration property leading
to deposition while for PEG assisted formulations the scenario dif-
fers because of the property of PEG which increases the instauration
leading to decreased deposition, though the difference in deposi-
tion does not indicate significant systemic toxicity [3,41,42].
4. Conclusions
The ability to obtain sustained in vitro and in vivo release of
andrographolide from the silica matrix was shown in this present
study. Based on the kinetic results, the faster release rate of andro-
grapholide during the initial stage (up to 6 h) and subsequent slow
release in the following stage (upto 168 h) were observed. Kinet-
ics of release showed the profile to be best fitted with zero-order
equation for the first 6 h, while for the rest of the period until the
168th hour, it obeyed Higuchi’s model. The intrinsic mechanism of
release predominantly followed the super case II transport for zero-
order while diffusion and erosion of the matrix for Higuchi’s model.
Almost 80% release accompanied with the highest burst release of
andrographolide was obtained only for the highest amount of PEG
(12 wt.%) in silica. Bioavailability of andrographolide from the sil-
ica carrier was found to be 168 h while in-case of crude material it
was seen to be nil after 7 h. Rapid increase in the drug plasma level
initially, followed by a steady state having significant correlation
with the in vitro biphasic release of the drug. Further, IVIVC can
also allow setting of more meaningful dissolution specifications.
The silica matrix became successful host for an extended period
without showing any toxic or degenerative effect.
Acknowledgments
We are grateful to Prof. Biswajit Mukherjee, Department of Phar-
maceutical Technology, Jadavpur University for providing space
for animal experiments. We are grateful and extend our heart-
felt gratitude to Dr. Munmun De for giving us assistance in animal
handling. Two of the authors (S.C & S.B) are thankful to the Uni-
versity Grants Commission for the financial assistance under the
scheme “Research Fellowship in Science for Meritorious Student”.
The author’s are grateful to “UGC-UPE” program for granting finan-
cial assistance in procurement of the HRTEM instrument. The
authors are thankful to the University Ethical Committee for grant-
ing permission for carrying out animal experiments.
Appendix A. Supplementary data
Supplementary material related to this article can be found,
in the online version, at http://dx.doi.org/10.1016/j.colsurfa.
2014.04.046.
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