Article

pubs.acs.org/Biomac

Curcumin Encapsulation in Submicrometer Spray-Dried Chitosan/

Tween 20 Particles
Martin G. O’Toole, Richard M. Henderson, Patricia A. Soucy, Brigitte H. Fasciotto, Patrick J. Hoblitzell,
Robert S. Keynton, William D. Ehringer, and Andrea S. Gobin*
Department of Bioengineering, University of Louisville, Louisville, Kentucky 40292, United States
*
S Supporting Information

ABSTRACT: Optimal curcumin delivery for medicinal

applications requires a drug delivery system that both
solubilizes curcumin and prevents degradation. To achieve
this, curcumin has been encapsulated in submicrometer
chitosan/Tween 20 particles via a benchtop spray-drying
process. Spray-drying parameters have been optimized using a
Taguchi statistical approach to minimize particle size and to
favor spheroid particles with smooth surfaces, as evaluated
with scanning electron microscopy (SEM) imaging. Nearly
spherical particles with 285 ± 30 nm diameter and 1.21 axial
ratio were achieved. Inclusion of curcumin in the spray-drying solution results in complete encapsulation of curcumin within the
chitosan/Tween 20 particles. Release studies confirm that curcumin can be released completely from the particles over a 2 h
period.

■ INTRODUCTION
Optimized drug delivery systems for curcumin, a major
component of turmeric extracted from the rhizome of Curcuma
longa, would offer new treatment options for a variety of
diseases.1 However, despite displaying potent antioxidant,2,3
anti-inflammatory,4,5 antiseptic,6 and analgesic7 activity, the
efficacy and widespread clinical use of curcumin is hampered by
poor bioavailability. Although high doses of curcumin have
been shown to be pharmacologically safe, oral administration of
curcumin is rapidly metabolized and excreted.8 Solubilization of
curcumin in aqueous solution typically requires addition of
organic solvents (e.g., methanol, dimethyl sulfoxide (DMSO))
or alkaline conditions. Unfortunately, curcumin quickly
decomposes under these conditions, with a pH-dependent
half-life on the order of seconds to minutes.9−11 The
insolubility and instability of curcumin thus provide a prime
opportunity for use of drug delivery agents to enhance
curcumin’s clinical relevance. To this effect, spray-dried
chitosan particles have been chosen as a potential drug delivery
vehicle to both solubilize and protect curcumin for medicinal
applications.
Chitosan, an amine-rich polysaccharide derived from chitin,
is well-known for its ability to form biodegradable and
biocompatible particulate systems for drug delivery.12−18
Chitosan has been shown to serve as a protective agent against
photolytic and hydrolytic degradation of curcumin.19 Chitosan-
based drug carriers for curcumin have also been shown to
protect curcumin from degradation, increase drug uptake, and
maintain therapeutic benefits.20 The carrier used in the previous
study, however, required several purification steps during
production and had a tendency to aggregate in solution.
Several methods are available for producing nanoparticles from
chitosan, including ionic gelation with tripolyphosphate,
microemulsion, emulsification solvent diffusion, polyelectrolyte
complexation, and spray-drying.12,21 Spray-drying offers the
advantage of producing a dry drug-loaded particle from a
polymer/drug solution in a single step. Several reports have
demonstrated the feasibility of spray-drying chitosan for drug
delivery formulations.21−34 However, typical particle sizes for
these methods have ranged from 1 to 50 μm. For intravenous
drug delivery applications, particle sizes should fall in the
submicrometer regime in order to traverse capillary networks
without causing embollism.35 Curcumin has also been
successfully formulated into drug delivery vehicles through
spray-drying with polymers such as ethyl cellulose, starch/
gelatin, malodextran, polyvinylpyrrolidone, and solid lipid
particles.33,36−41 However, there are little if any available
accounts of encapsulating curcumin in chitosan particles via
spray-drying.
A recent advance for producing submicrometer-sized
particles via spray-drying on a laboratory scale is the
commercial availability of the Buchi B-90 Nanospray spray
dryer.42,43 During the spray-drying process with the B-90, a
polymer solution is sonicated and forced through a fine (4−7
μm) mesh into a heated air stream. Particles are formed from
the aerosolized polymer during flight down a heated column
during which the solvent rapidly evaporates. Addition of small
molecules to the spray-drying mixture allows for encapsulation
Received: April 11, 2012
Revised: June 26, 2012
Published: June 27, 2012

© 2012 American Chemical Society 2309 dx.doi.org/10.1021/bm300564v | Biomacromolecules 2012, 13, 2309−2314
Biomacromolecules
within the polymeric particle framework. The dry particles are
then collected onto a charged precipitation drum at the end of
the airshaft. Several user-defined variables impact the resultant
size and morphology of the spray-dried particles, including
solvent composition, polymer concentration, surfactant con-
centration, spray-drying temperature, and rate of airflow
through the spray dryer. Proper optimization of the spray-
drying parameters can achieve submicrometer particle sizes.
In this study, we examined the ability of curcumin to be
encapsulated into submicrometer-sized chitosan particles for
use in drug delivery. Tween 20 has been incorporated into the
formulation as a solubilizing agent for curcumin.44,45 Spray-
drying conditions have been optimized for small particle size
and spherical morphology using Taguchi statistical analysis of
measurements from scanning electron microscopy (SEM)
images. Naturally fluorescent curcumin and fluorescently
labeled Tween 20 have been utilized to image the particles
with incorporated surfactant using confocal microscopy.
Curcumin stability after release from particles was determined
via spectroscopic analysis. Our findings suggest that these
particles will provide a platform to overcome current problems
associated with curcumin delivery. The bioactivity of curcumin,
once released from the particles, will be the subject of a future
report.
■
MATERIALS AND METHODS
Chemical Supplies. Chitosan 90/200 was purchased from Heppe
Medical Chitosan, GmbH (Halle, Germany). Curcumin (85% pure;
demethoxycurcumin and bis-demethoxycurcumin as impurities), acetic
acid, 1-octanol, ascorbic acid, 3,5-di-tert-butyl-4-hydroxytoluene
(BHT), carbonyldiimidazole (CDI), dichloromethane, diethyl ether,
DMSO, polyethylene glycol sorbitan monolaurate (Tween 20), and
polyethylene glycol sorbitan monooleate (Tween 80) were purchased
from Sigma Aldrich (St. Louis, MO, USA). Magnesium sulfate was
purchased from Fisher Scientific (Pittsburgh, PA USA). Texas Red
Hydrazide and SlowFade antifade reagent were purchased from
Invitrogen Corporation (Carlsbad, CA, USA).
Curcumin Saturation Studies. Curcumin (50 mg) was added to
100 mL 1% acetic acid containing 0−0.05 w/v% Tween 20 with or
without 0.05 w/v% chitosan. The solution was stirred for 1 h in the
dark followed by centrifugation (3489g, 5 min). The supernatant was
decanted and the UV/visible spectrum collected on a Beckmann
Coulter DB-525 spectrometer (Brea, CA). The absorbance at 425 nm
was used to quantify curcumin based on calibrations made with known
amounts of curcumin dissolved in 1% acetic acid with 0.1 w/v%
Tween 20.
Synthesis of Texas Red-Labeled Tween 20. CDI-activated
Tween 20 was prepared according to published protocols.46,47 The
procedure for preparation of Texas Red-labeled Tween 20 from CDI-
activated Tween 20 is a modification of the procedure published by
Yoshimura, et al.48 Care was taken during all manipulations to
minimize light exposure. CDI-activated Tween 20 (6 mg, 4.8 μmol)
was dissolved in 25 mL phosphate-buffered saline (PBS) buffer with 3
mg (4.8 μmol) of Texas Red Hydrazide (Invitrogen). The reaction was
stirred for 2 h at room temperature. The dark red product was
extracted with 3 × 50 mL dichloromethane, dried over MgSO4, gravity
filtered, and the solvent was removed by rotary evaporation leaving a
bright red oily product.
Determination of Detergents for Spray-Drying Formulation.
Chitosan-based particles were produced using a Buchi B-90 Nanospray
spray dryer (New Castle, DE). Solutions 0.05 w/v% chitosan with or
without 0.025 w/v% detergent (Tween 20 or Tween 80) were stirred
overnight and filtered through a 0.45 μm syringe filter before spray-
drying. Solutions were spray-dried through a 4 μm spray mesh using
an inlet temperature of 120 °C and air flow rate of 150 L/min. Dry
particles were collected from the collecting drum using a particle
scraper and stored in a desiccator.
2310
Article
Chitosan-Based Particle Production via Spray-Drying.
Chitosan-based particles were produced using a Buchi B-90 Nanospray
spray dryer (New Castle, DE). Solutions of various concentrations of
chitosan and detergent were stirred overnight and filtered through a
0.45 μm syringe filter before spray-drying. Solutions of polymer with
and without detergent or curcumin were spray-dried through a 4 μm
spray mesh using various combinations of the following parameters:
Inlet temperature = 80, 100, or 120 °C; air flow rate = 100, 120, or 150
L/min; chitosan concentration = 0.025, 0.05, or 0.1 w/v%; Tween 20
concentration = 0, 0.025, or 0.05 w/v%. Specific combinations of
parameters were determined using a Taguchi array. Dry particles were
collected from the collecting drum using a particle scraper and stored
in a desiccator.
Particle Characterization. Spray-dried particles were character-
ized using a Zeiss Supra 35 (Carl Zeiss Microscopy, LLC, Thornwood,
NY) field emission scanning electron microscope. Samples were
prepared by placing dry particles onto sample pedestals coated with
silver paint followed by coating with gold/palladium alloy for 30 s
using a plasma coater. Images were captured using EHT = 2 kV.
Particle diameter and axial ratio measurements were determined using
ImageJ software on randomly selected particles (150 particles/image)
from three separate SEM images for each sample (n = 3 samples for
each spray-drying condition; 1350 particles for each condition).
Particle diameter was based on the longest particle dimension. The
axial ratio is defined as the ratio of the largest particle dimension to the
smallest. Particle morphology is determined from the axial ratio
measurement, with spherical defined as an axial ratio of 1. Statistical
comparison of size was performed using Student t tests with p ≤ 0.05
considered significantly different.
Statistical Analysis of Particle Size and Morphology.
Optimization of spray-drying parameters was completed using the
Taguchi-based statistical method, which is designed for optimizing a
large number of parameters using the fewest number of trials.42,49,50
Parameters to be optimized were chitosan concentration, detergent
concentration, spray dryer inlet temperature, and airflow rate. Each
parameter was evaluated at three different levels (Table S1). An L9
orthogonal array (four parameters, three parameter levels, nine
experiments) was used to determine the instrumental parameters.
Design criteria for particle size and morphology were to produce the
smallest particles that were as close to spherical as possible. Parameter
optimization was quantified by maximizing the experimental signal-to-
noise (S/N) ratio using the formula:
i=1
(S/N)i = − 10·log10[∑ (yi2 /n)]
n
where y is the value returned for the parameter being optimized, and n
is the number of experimental trials. Particle diameters for each
condition were compared.
Confocal Imaging. Spray-dried particles were imaged on a Nikon
A1 laser scanning confocal microscope (Melville, NY). Samples were
prepared by placing a small amount of dried particles (with or without
curcumin and with or without Texas Red-labeled Tween 20) on a
microscope slide followed by a few drops of SlowFade antifade
reagent. A glass coverslip was then affixed over the sample. Curcumin
fluorescence was monitored on the green channel (Ex/Em = 488/525
nm), and Texas Red was monitored on the red channel (Ex/Em =
561/595 nm)
Curcumin Release Studies. Curcumin or curcumin encapsulated
in chitosan/Tween 20 particles (2 mg/mL) was placed in 1% acetic
acid or 1× PBS buffer with 1% ascorbic acid as preservative at 37 °C in
the dark.51 Curcumin release from the particles was evaluated by
partitioning a small aliquot of the release mixture with 1-octanol
containing 0.1% BHT as preservative at various time points. The
aqueous/organic mixture was centrifuged for 5 min at 218g to aid in
separation of layers. The organic layer was removed, and curcumin
content was evaluated by measuring the absorbance at 425 nm.
dx.doi.org/10.1021/bm300564v | Biomacromolecules 2012, 13, 2309−2314
Biomacromolecules
■
RESULTS AND DISCUSSION
Detergent Choice and Solubilizing Curcumin. Due to
the poor solubility of curcumin in 1% acetic acid solution,
fabrication of curcumin chitosan particles via spray-drying was
not successful. To improve curcumin’s solubility, Tween 20 and
Tween 80 were screened as potential solubilizing agents based
on known biocompatibility and previous successful use in
spray-drying experiments.42 Chitosan (0.05 w/v%) was
prepared for spray-drying by filtering through a 0.45 μm filter
after stirring in 0.025 w/v% Tween 20 or Tween 80 in an acetic
acid solution overnight. Samples of chitosan were spray-dried
through a 4 μm mesh with and without the presence of Tween
20 or Tween 80 using a chitosan concentration of 0.05 w/v%,
0.025 w/v% detergent, 120 °C inlet temperature, and 150 L/
min flow rate.42 SEM analysis of the resulting particles showed
that average particle size for samples with Tween 20, Tween 80,
and samples without detergent were not statistically different
(360 ± 164, 325 ± 145, and 346 ± 37 nm, respectively; Figure
1a). The surfaces of the particles spray-dried without detergent
Figure 1. SEM images of chitosan particles spray-dried in the presence
of (a) Tween 20 (T20), (b) Tween 80 (T80), and (c) no surfactant
(NS). Size analysis results are displayed in bar graph form (d). Spray-
drying conditions: chitosan concentration = 0.05 w/v%, surfactant
concentration = 0.025 w/v%, inlet temperature = 120 °C, airflow =
150 L/min.
or with Tween 20 appeared smooth, whereas the samples
spray-dried with Tween 80 displayed “pitted” surfaces. Tween
20 was chosen for inclusion in future spray-drying experiments,
as it did not detrimentally affect the particle size and produced
particles with comparatively smooth surfaces (Figure 1).
The ability of Tween 20 to solubilize curcumin in 1% acetic
acid was evaluated by measuring the curcumin concentration in
curcumin-saturated acetic acid solutions with varying amounts
of Tween 20. The acidic nature of these solutions allowed for
prolonged monitoring of curcumin with minimal degradation.
Solubility results were compared with identical solutions that
also contained 0.05 w/v% chitosan (Figure 2). The curcumin
saturation point increases linearly with Tween 20 concentration
throughout the range used in these experiments, with an upper
limit of 294 μM curcumin with 0.05 w/v% Tween 20. This
represents a 12.7-fold increase in curcumin solubility. At each
Tween 20 concentration, the presence of chitosan in the
solution increases the curcumin saturation point by an average
2311
Article
Figure 2. Solubility of curcumin in 1% acetic acid with increasing
concentration of Tween 20 displayed with no chitosan added (gray
bars) and with 0.05 w/v% chitosan (white bars). Curcumin
concentration monitored at 420 nm in saturated solutions.
of 19.6%. The ability of chitosan to help solubilize curcumin has
been attributed to hydrogen bonding interactions between
chitosan −NH2 and curcumin phenol −OH groups.19
Spray-Drying Optimization. Four spray-drying parame-
ters were chosen for optimization (Table S1). All samples were
sprayed through a 4 μm spray head mesh, as the 5.5 and 7 μm
meshes provided with the spray dryer produced particles that
were deemed too large for circulatory system drug delivery
(data not shown). Chitosan concentration levels were restricted
to 0.1 w/v% or less to avoid clogging the spray head. Table S2
lists the Taguchi orthogonal array utilized for this experiment as
well as the resulting S/N ratios calculated from the resulting
particle size and axial ratio data. The smallest particle sizes (285
± 30 nm) were produced in trial 9, using a chitosan
concentration of 0.025 w/v%, no Tween 20, 120 °C inlet
temperature, and 120 L/min flow rate. The most spherical
particles (axial ratio = 1.12) were produced using the
parameters for trial 2: chitosan concentration of 0.05 w/v%,
Tween 20 concentration of 0.5 w/v%, 120 °C inlet temper-
ature, and 100 L/min.
By averaging all of the trial results for each level of a given
parameter (e.g., all of the trials using a chitosan concentration
of 0.05 w/v%), it is possible to determine which parameter had
the greatest impact on particle size and morphology. The
impact of each parameter is ranked according to the degree of
variance of the experimental result obtained from varying that
parameter (Tables S3 and S4). Both particle size and
morphology were impacted most by choice of chitosan
concentration, with the smallest and most spheroid particles
being produced with the lowest chitosan concentration. Particle
size was also greatly impacted by airflow rate, although not in a
reliably predictive manner. Representative SEM images of the
particles showing the effect of chitosan concentration on
particle size are displayed in Figure 3. Note that with the high
concentrations of chitosan, a bimodal distribution of particle
size occurs. As samples spray-dried with low chitosan
concentrations had unimodal size distributions, the standard
deviation of these trials were markedly lower (±30 nm versus
±569 nm for the bimodal high concentration trials).
Although in preliminary trials particles produced with and
without Tween 20 had smooth surfaces, during the
optimization trials samples tended to yield more particles
with a wrinkled appearance (Figure 3c). This effect was more
substantial with higher chitosan concentrations. When using 0.1
dx.doi.org/10.1021/bm300564v | Biomacromolecules 2012, 13, 2309−2314
Biomacromolecules
Figure 3. SEM images of chitosan particles spray-dried with (a) 0.025
w/v% chitosan and 0 w/v% Tween 20, (b) 0.05 w/v% chitosan and
0.025 w/v% Tween 20, (c) 0.1 w/v% chitosan and 0.025 w/v% Tween
20, and (d) 0.05 w/v% chitosan and 0.05 w/v% Tween 20.
w/v% chitosan and no detergent, 20−50% of the particles
displayed a wrinkled appearance. When the chitosan concen-
tration was lowered to 0.05 w/v%, only 5−10% of the particles
had wrinkled surfaces. Several incompletely formed particles
were found during SEM analysis. These particles contained
large holes in the polymer shell that allow confirmation that the
particles are hollow, as is typical for spray-dried particles
(Figure 3d).
While the spray-drying optimization results can be used to
produce either the smallest or most spherical particles, these
attributes alone may not be the most important to consider
when choosing an optimum set of conditions for encapsulation
of curcumin. For example, the parameters that produced the
smallest particles were from trial 9: 0.025 w/v% chitosan, 0
Tween 20, 120 °C inlet temperature and 120 L/min airflow.
These parameters involved no detergent, which in our
preliminary studies was deemed necessary for solubilizing
curcumin in the spray-drying solution, and therefore makes a
poor set of conditions for curcumin encapsulation. Additionally,
the use of only 0.025 w/v% chitosan resulted in much longer
spray-drying times required for producing an adequate amount
of material. The most spherical particles were produced in trial
2, using 0.05 w/v% chitosan, 0.05 w/v% Tween 20, 120 °C, and
100 L/min air flow. This trial included equal amounts, weight
for weight, of chitosan and Tween 20. Comparing results of
trial 2 with trials using less Tween 20 reveals that decreased
Tween 20 has little detriment to particle shape. Additionally,
this trial yielded only the fifth smallest batch of particles.
Therefore, in the interest of minimizing both particle size and
the amount of “auxiliary” components in the spray-drying
mixture, this trial was rejected as a candidate for curcumin
encapsulation.
The ideal set of conditions for encapsulating curcumin via
spray-drying would therefore produce small, nearly spherical
particles using a minimum amount of Tween 20 to solubilize
curcumin and a chitosan concentration large enough to reduce
spray-drying times. Perusing the data presented in Table S2
(Supporting Information) shows that the conditions and results
from trial 1 meet all of these criteria. The conditions of trial 1
(0.05 w/v% chitosan, 0.025 w/v% Tween 20, 150 °C, and 120
2312
Article
L/min air flow) produced the second smallest batch of particles
with an axial ratio that was within 5% of that obtained for trial 2
(the condition producing the most spherical particles).
Curcumin Encapsulation. Curcumin was spray-dried
using the parameters from chitosan trial 1. UV−visible
spectroscopy was used to probe for curcumin decomposition
due to the spray-drying process (Figure 4). Curcumin
Figure 4. UV/visible spectrum of curcumin before (solid trace) and
after (dashed trace) spray-drying in 1% acetic acid with 0.025 w/v%
Tween 20 and 0.05 w/v% chitosan. Spray-dried at 120 °C with 150 L/
min air flow.
decomposition is easily monitored by a decrease in intensity
of the peak at 425 nm in concert with a broadening of the
absorption band and appearance of two new peaks at 365 and
335 nm due to degradation of curcumin to trans-6-(4′-hydroxy-
3′-methoxyphenyl)-2,4-dioxo-5-hexenal.52 Despite the high
temperature of the spray head (150 °C) and sonication, the
UV−visible spectrum of curcumin after spray-drying is identical
in content to that of the same solution before spray-drying. An
8.5 ± 0.6% (n = 3) reduction in peak intensity at 425 nm
represents minimal loss due to the spray-drying process. To
determine the effect of curcumin loading on particle size,
particles were spray-dried using the previously determined
optimized conditions with the inclusion of various curcumin
concentrations below the saturation point (Table S5). SEM
measurements reveal that encapsulation of curcumin results in a
nearly linear increase in chitosan particle diameter. Attempts to
extract curcumin from the dry particles with repeated ethyl
acetate washings with vigorous shaking resulted in no
detectable traces of curcumin in the ethyl acetate, implying
that all curcumin in the sample is trapped within the chitosan
particles. From this it is determined that curcumin
encapsulation efficiency for this process is nearly 100%.53 It is
therefore assumed that the increase in particle size in these
experiments is due to curcumin loading.
Particle Composition. The fate of Tween 20 present in the
spray-drying mixture was determined through spray-drying
particles with Tween 20 covalently modified with Texas Red
fluorescent dye.48 Confocal microscopy shows that the Tween
20 dye is incorporated into the chitosan particle structure and
tends to be concentrated on the outer periphery of the
particles, suggesting that the detergent is accumulated around
the chitosan shell, most likely due to hydrogen bonding
interactions (Figure 5a).54 The naturally occurring fluorescence
of curcumin can be used to visualize its compartmentalization
within the particles as well (Figure 5b). Unlike the Texas Red−
Tween 20, the curcumin appears to be evenly distributed
throughout the particle. An overlay of both fluorescence images
dx.doi.org/10.1021/bm300564v | Biomacromolecules 2012, 13, 2309−2314
Biomacromolecules
Figure 5. Confocal microscopy images (single xy plane of a z-stack) of curcumin-loaded chitosan/Tween 20 particles: (a) Tween 20−Texas Red
shown in red, (b) curcumin shown in green, (c) overlay of Texas Red and FITC channels, and (d) 10× zoom of overlay of Texas Red and FITC
channels of curcumin-loaded chitosan/Texas Red-labeled Tween 20 particles. Scale bar = 10 μm.
displays the curcumin and Tween 20 spatial distribution within
the particles (Figure 5c,d, respectively).
Curcumin Release Studies. Curcumin release studies were
conducted in both 1% acetic acid and 1× PBS buffer to ensure
dissolution of the chitosan/Tween 20 particles. The release
medium was fortified with 1% ascorbic acid and the organic
layer with 0.1% BHT as preservatives.51 Release studies in the
presence of preservatives in either solvent show a burst release
profile for curcumin, with nearly 40% of curcumin released in
the first 5 min of the experiment (Figure 6). All detectable
curcumin release was complete within 2 h (Figure 6).
■ CONCLUSIONS
The feasibility of encapsulating curcumin in submicrometer
chitosan particles via spray-drying has been demonstrated. The
Figure 6. Release profile of curcumin from chitosan/Tween 20
particles in 1% acetic acid (⧫) and PBS buffer (○) (n = 3).
2313
Article
spray-drying process has been optimized for encapsulation of
curcumin in the smallest, most spherical particles possible with
the smoothest morphology. Release studies confirm a burst
release profile for curcumin. Future studies will involve chitosan
derivatives that display better solubility under physiological
conditions for applications in systemic drug delivery. In
addition, the use of cross-linking agents will be explored for
altering curcumin release kinetics under physiologic conditions.
The incorporation of preservative molecules in addition to
curcumin within the chitosan capsules will also be explored.
■
*
ASSOCIATED CONTENT
S Supporting Information
Tables S1−S5 are included in the Supporting Information for
this manuscript. This material is available free of charge via the
Internet at http://pubs.acs.org.
■ AUTHOR INFORMATION
Corresponding Author
*Tel: 502 852 8321. Fax: 502 852 6806. E-mail: andrea.gobin@
louisville.edu.
Notes
The authors declare no competing financial interest.
■ ACKNOWLEDGMENTS
Support for this work was in part provided by NASA Grant
NNX10AJ36G. The authors wish to acknowledge Joe Williams
and Dhruvinkumar Patel for their assistance with SEM imaging.
dx.doi.org/10.1021/bm300564v | Biomacromolecules 2012, 13, 2309−2314
Biomacromolecules
■
REFERENCES
(1) Aggarwal, B. B.; Surh, Y.-J.; Shishodia, S. The Molecular Targets
and Therapeutic Uses of Curcumin in Health and Disease; Springer: New
York, 2007.
(2) Motterlini, R.; Foresti, R.; Bassi, R.; Green, C. J. Free Radical Biol.
Med. 2000, 28, 1303−1312.
(3) Kuo, M. L.; Huang, T. S.; Lin, J. K. Biochim. Biophys. Acta, Mol.
Basis Dis. 1996, 1317, 95−100.
(4) Kawamori, T.; Lubet, R.; Steele, V. E.; Kelloff, G. J.; Kaskey, R.
B.; Rao, C. V.; Reddy, B. S. Cancer Res. 1999, 59, 597−601.
(5) Srimal, R. C.; Dhawan, B. N. J. Pharm. Pharmacol. 1973, 25, 447−
452.
(6) De, R.; Kundu, P.; Swarnakar, S.; Ramamurthy, T.; Chowdhury,
A.; Nair, G. B.; Mukhopadhyay, A. K. Antimicrob. Agents Chemother.
2009, 53, 1592−1597.
(7) Agarwal, K. A.; Tripathi, C. D.; Agarwal, B. B.; Saluja, S. Surg.
Endosc. 2011, 25, 3805−3810.
(8) Anand, P.; Kunnumakkara, A. B.; Newman, R. A.; Aggarwal, B. B.
Mol. Pharmacol. 2007, 4, 807−818.
(9) Tonnesen, H. H.; Karlsen, J. Z. Lebensm. Unters. Forsch. 1985,
180, 402−404.
(10) Wang, Y. J.; Pan, M. H.; Cheng, A. L.; Lin, L. I.; Ho, Y. S.;
Hsieh, C. Y.; Lin, J. K. J. Pharm. Biomed. Anal. 1997, 15, 1867−1876.
(11) Blasius, R.; Duvoix, A.; Morceau, F.; Schnekenburger, M.;
Delhalle, S.; Henry, E.; Dicato, M.; Diederich, M. Ann. N.Y. Acad. Sci.
2004, 1030, 442−448.
(12) Nagpal, K.; Singh, S. K.; Mishra, D. N. Chem. Pharm. Bull.
(Tokyo) 2010, 58, 1423−1430.
(13) Akhtar, F.; Kar, S. J. Biotechnol. 2010, 150, S93−S93.
(14) Aktas, Y.; Andrieux, K.; Alonso, M. J.; Calvo, P.; Gursoy, R. N.;
Couvreur, P.; Capan, Y. Int. J. Pharm. 2005, 298, 378−383.
(15) Anitha, A.; Deepagan, V. G.; Rani, V. V. D.; Menon, D.; Nair, S.
V.; Jayakumar, R. Carbohydr. Polym. 2011, 84, 1158−1164.
(16) Carvalho, E. L. S.; Grenha, A.; Remuñań -López, C.; Alonso, M.
J.; Seijo, B. Methods Enzymol. 2009, 465, 289−312.
(17) Gaspar, V. M.; Sousa, F.; Queiroz, J. A.; Correia, I. J.
Nanotechnology 2011, 22, 015101.
(18) Prego, C.; Torres, D.; Fernandez-Megia, E.; Novoa-Carballal, R.;
Quinoa, E.; Alonso, M. J. J. Controlled Release 2006, 111, 299−308.
(19) Mitra, S. P. J. Surface. Sci. Technol. 2008, 24, 39−55.
(20) Akhtar, F.; Rizvi, M. M.; Kar, S. K. Biotechnol. Adv. 2012, 30,
310−320.
(21) He, P.; Davis, S. S.; Illum, L. Int. J. Pharm. 1999, 187, 53−65.
(22) Cerchiara, T.; Luppi, B.; Bigucci, F.; Zecchi, V. J. Pharm.
Pharmacol. 2003, 55, 1623−1627.
(23) Cervera, M. F.; Heinamaki, J.; de la Paz, N.; Lopez, O.; Maunu,
S. L.; Virtanen, T.; Hatanpaa, T.; Antikainen, O.; Nogueira, A.;
Fundora, J.; Yliruusi, J. AAPS PharmSciTech 2011, 12, 637−649.
(24) Corrigan, D. O.; Healy, A. M.; Corrigan, O. I. Eur. J. Pharm.
Biopharm. 2006, 62, 295−305.
(25) Harikarnpakdee, S.; Lipipun, V.; Sutanthavibul, N.; Ritthidej, G.
C. AAPS PharmSciTech 2006, 7, E12−E17.
(26) Muzzarelli, C.; Tosi, G.; Francescangeli, O.; Muzzarelli, R. A. A.
Carbohydr. Res. 2003, 338, 2247−2255.
(27) Nunthanid, J.; Huanbutta, K.; Luangtana-Anan, M.;
Sriamornsak, P.; Limmatvapirat, S.; Puttipipatkhachorn, S. Eur. J.
Pharm. Biopharm. 2008, 68, 253−259.
(28) Nunthanid, J.; Laungtana-Anan, A.; Sriamornsak, P.;
Limmatvapirat, S.; Puttipipatkhachorn, S.; Lim, L. Y.; Khor, E. J.
Controlled Release 2004, 99, 15−26.
(29) Rege, P. R.; Garmise, R. J.; Block, L. H. Int. J. Pharm. 2003, 252,
41−51.
(30) Shi, X.; Tan, T. J. Biomed. Eng./Shengwu Yixue Gongchengxue
Zazhi 2003, 20, 26−29.
(31) Takeuchi, H.; Yasuji, T.; Yamamoto, H.; Kawashima, Y. Pharm.
Res. 2000, 17, 94−99.
(32) Weerakody, R.; Fagan, P.; Kosaraju, S. L. Int. J. Pharm. 2008,
357, 213−218.
2314
Article
(33) Duan, J. H.; Zhang, Y. D.; Han, S. W.; Chen, Y. X.; Li, B.; Liao,
M. M.; Chen, W.; Deng, X. M.; Zhao, J. F.; Huang, B. Y. Int. J. Pharm.
2010, 400, 211−220.
(34) Liu, W.; Wu, W. D.; Selomulya, C.; Chen, X. D. Int. J. Chem.
Eng. 2011, 267218.
(35) Torchilin, V. P. Nanoparticulates as Drug Carriers; Imperial
College Press: London/Hackensack, NJ, 2006.
(36) Donsi, F.; Wang, Y. W.; Li, J.; Huang, Q. R. J. Agric. Food Chem.
2010, 58, 2848−2853.
(37) Gogu, P. K.; Jithan, A. V. Ind. J. Pharm. Sci. 2010, 72, 346−352.
(38) Kshirsagar, A. C.; Yenge, V. B.; Sarkar, A.; Singhal, R. S. Food
Chem. 2009, 113, 1139−1145.
(39) Paradkar, A.; Ambike, A. A.; Jadhav, B. K.; Mahadik, K. R. Int. J.
Pharm. 2004, 271 (1−2), 281−286.
(40) Wang, Y.; Lu, Z.; Lv, F.; Bie, X. Eur. Food Res. Technol. 2009,
229 (3), 391−396.
(41) Zhang, F.; Koh, G. Y.; Jeansonne, D. P.; Hollingsworth, J.;
Russo, P. S.; Vicente, G.; Stout, R. W.; Liu, Z. J. J. Pharm. Sci. 2011,
100 (7), 2778−2789.
(42) Lee, S. H.; Heng, D.; Ng, W. K.; Chan, H.-K.; Tan, R. B. H. Int.
J. Pharm. 2011, 403 (1−2), 192−200.
(43) Li, X.; Anton, N.; Arpagaus, C.; Belleteix, F.; Vandamme, T. F. J.
Controlled Release 2010, 147 (2), 304−310.
(44) Xiao, D.; Gommel, C.; Davidson, P. M.; Zhong, Q. X. J. Agric.
Food Chem. 2011, 59 (17), 9572−9580.
(45) Gowthamarajan, K.; Singh, S. K. Dissolution Technol. 2010, 17
(3), 24−32.
(46) Dai, H. J.; Chen, R. J.; Bangsaruntip, S.; Drouvalakis, K. A.;
Kam, N. W. S.; Shim, M.; Li, Y. M.; Kim, W.; Utz, P. J. Proc. Natl.
Acad. Sci. U.S.A. 2003, 100 (9), 4984−4989.
(47) Hermanson, G. T. Bioconjugate Techniques, 2nd ed.; Academic
Press: San Diego, CA, 1996.
(48) Yoshimura, S. Y., S. H.; Khan, S.; Maruyama, H.; Nakayama, Y.;
Takeyasu, K. Biomacromolecules 2011, 12 (4), 1200−1204.
(49) Diemunsch, A. M.; Pabst, J. Y.; Constant, C.; Mathis, C.;
Stamm, A. Drug Dev. Ind. Pharm. 1993, 19 (12), 1461−1477.
(50) Kim, H. T.; Kim, K. D.; Choi, D. W.; Choa, Y. H. Colloids Surf.,
A 2007, 311 (1−3), 170−173.
(51) Jithan, A. V.; Madhavi, K.; Madhavi, M.; Prabhakar, K. Int. J.
Pharm. Invest. 2011, 1 (2), 119.
(52) Sundaryono, A.; Nourmamode, A.; Gardrat, C.; Grelier, S.;
Bravic, G.; Chasseau, D.; Castellan, A. Photochem. Photobiol. Sci. 2003,
2 (9), 914−920.
(53) Das, R. K.; Kasoju, N.; Bora, U. Nanomed.: Nanotechnol., Biol.
Med. 2010, 6 (1), 153−160.
(54) Ziani, K.; Oses, J.; Coma, V.; Mate, J. I. LWTFood Sci.
Technol. 2008, 41 (10), 2159−2165.
dx.doi.org/10.1021/bm300564v | Biomacromolecules 2012, 13, 2309−2314