Release-Modulated Antioxidant Activity of a Composite Curcumin-Chitosan Polymer

Article abstract of DOI:10.1021/acs.biomac.5b01019

Curcumin is known to have immense therapeutic potential but is hindered by poor solubility and rapid degradation in solution. To overcome these shortcomings, curcumin has been conjugated to chitosan through a pendant glutaric anhydride linker using amide

Full text of DOI:10.1021/acs.biomac.5b01019

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Article
Release-Modulated Antioxidant Activity of
a Composite Curcumin-chitosan Polymer
Martin G O'Toole, Patricia A Soucy, Rajat Chauhan, Mandapati V. R. Raju,
Dhruvina Nayankumar Patel, Betty Marie Nunn, Megan A Keynton, William D
Ehringer, Michael H. Nantz, Robert S Keynton, and Andrea Samantha Gobin
Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/acs.biomac.5b01019 • Publication Date (Web): 23 Feb 2016
Downloaded from http://pubs.acs.org on February 27, 2016
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Release-Modulated Antioxidant Activity of a
Composite Curcumin-chitosan Polymer
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Martin G. O’Toole1,*, Patricia A. Soucy1, Rajat Chauhan1, Mandapati V. R. Raju2,
Dhruvina N. Patel1, Betty M. Nunn1, Megan A. Keynton1, William D. Ehringer1, Michael
H. Nantz2, Robert S. Keynton1, Andrea S. Gobin1
1Department of Bioengineering, and 2 Department of Chemistry, University of Louisville,
Louisville, Kentucky 40292, United States
KEYWORDS Curcumin; Chitosan; Biopolymer; Covalent Modification; Antioxidant
modulation; Drug release
Curcumin is known to have immense therapeutic potential but is hindered by poor
solubility and rapid degradation in solution. To overcome these shortcomings, curcumin
has been conjugated to chitosan through a pendant glutaric anhydride linker using amide
bond coupling chemistry. The hybrid polymer has been characterized by UV-visible,
fluorescence and infrared spectroscopies as well as zeta potential measurements and SEM
imaging. The conjugation reactivity was confirmed through gel permeation
chromatography and quantification of un-conjugated curcumin. An analogous reaction of
curcumin with glucosamine, a small molecule analog for chitosan was performed and the
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purified product characterized by mass spectrometry, UV-visible, fluorescence, and
infrared spectroscopies. Conjugation of curcumin to chitosan has greatly improved
curcumin aqueous solubility and stability, with no significant curcumin degradation
detected after one month in solution. The absorbance and fluorescence properties of
curcumin are minimally perturbed (λmax shifts of 2 nm and 5 nm, respectively) by the
conjugation reaction. This conjugation strategy required use of one out of two curcumin
phenols (one of the main antioxidant functional groups) for covalent linkage to chitosan,
thus temporarily attenuating its antioxidant capacity. Hydrolysis-based release of
curcumin from the polymer, however, is accompanied by full restoration of curcumin’s
antioxidant potential. Antioxidant assays show that curcumin radical scavenging potential
is reduced by 40% after conjugation, but that full antioxidant potential is restored upon
hydrolytic release from chitosan. Release studies show that curcumin is released over 19
days from the polymer and maintains a concentration of 0.23 0.12 ꢀM curcumin/mg
polymer/mL solution based on 1% curcumin loading on the polymer. Release studies in
the presence of carbonic anhydrase, an enzyme with known phenolic esterase activity,
show no significant difference from non-enzymatic release studies, implying that simple
ester hydrolysis is the dominant release mechanism. Conjugation of curcumin to chitosan
through a phenol ester modification provides improved stability and solubility to
curcumin, with ester hydrolysis restoring the full antioxidant potential of curcumin.
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Introduction
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Oxidative stress is characterized by an imbalance between the production of reactive
oxygen species (ROS) and a biological system's ability to readily remove the reactive
intermediates or easily repair the subsequent damage.1 The resulting indiscriminate
damage caused by ROS can lead to severe metabolic dysfunctions, loss of cell integrity,
hindrance of enzyme function, genomic instability, and other detrimental effects.
Oxidative stress at the cellular level can ultimately lead to pathogenesis of many human
disease states, such as inflammation, ischemia, atherosclerosis, arthritis, cancer,
Parkinson’s disease, aging and Alzheimer’s disease.2 To counteract oxidative stress, the
body produces an armory of antioxidants to defend itself. These complex antioxidant
systems, consisting of antioxidant enzymes and small antioxidant molecules, act in
concert to decrease the ROS load. However, during times of excess ROS production,
whether inspired by biological processes or external stimuli, such as acute radiation
exposure, it is beneficial to supplement natural antioxidant capacities through therapeutic
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A)
antioxidant agents. It is therefore
desirable to seek new methods for
bolstering
the
body’s
native
antioxidant capacity.
B)
Curcumin (diferuloylmethane) is a
polyphenol extract from the rhizome of
the Curcuma Longa herb from which
the culinary spice, turmeric, is derived,
Figure 1. Chemical structures of curcumin
(A) and chitosan (B).
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(Figure 1A). It is a major component of turmeric that displays an intense yellow color and
naturally fluoresces in the green spectrum.2 The potent antioxidant activity of curcumin is
provided by its diketone and phenol moieties that are known to scavenge free radicals.3
Curcumin is known to interact with a variety of transcription factors, growth factors and
their receptors, cytokines, enzymes, and genes. Specifically in the biological field of
tumorigenesis, curcumin is a potent inhibitor of nuclear factor-κB (NF-κB), a
transcription factor implicated in pathogeneses of several malignancies; inhibits the
production of various angiogenic cytokines,2, 4 and enhances the expression of the tumor
suppressor protein p53 and its downstream target p21CIP1/WAF1.5 Curcumin also has
potent anti-inflammatory and wound healing properties. Curcumin quenches free radicals
and reduces inflammation through NF-κB, COX-2, LOX, and iNOS inhibition.3a, 6
However, the effectiveness of curcumin in vivo is limited by low solubility in aqueous
solution and rapid degradation.7 Alkaline hydrolysis is the main process involved in the
degradation of curcumin in buffer solutions, depleting 90% of curcumin in 30 minutes at
physiological conditions.8 Many analogues of curcumin have been created to increase its
solubility, but they typically have reduced potency.5a, 9 To improve the bioavailability of
curcumin, it has been encapsulated in synthetic liposomes, polymeric nanoparticles,
microspheres, and hydrogels, all of which demonstrated low residence time/ fast release
kinetics.10 In order to increase the release time for curcumin, we have been exploring
covalent conjugation strategies that incorporate bio-degradable linkages. However, the
three chemical functionalities on curcumin that most readily lend themselves to
conjugation reactions, the two phenol moieties and the central diketone, are major
components of curcumin’s antioxidant potential.3a There is, therefore, a high probability
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that covalent conjugation of curcumin to a delivery vehicle is concomitant with a partial
loss of antioxidant potential. Therefore, a delivery system that enhances the solubility of
curcumin, protects it from degradation, and retains its antioxidant properties could
improve its bioavailability and effectiveness. As such, from our previous work
encapsulating curcumin within polymer shells, it seemed feasible that a composite
polymer of curcumin and chitosan could provide many of these enhanced properties.11
Chitosan (CS), a cationic polymer whose crystallinity is a function of the degree of
deacetylation (range = 50–95%), is derived from chitin, an abundant polysaccharide
found in crustacean shells (Figure 1B). CS has been shown to display wound-healing
properties, is nontoxic, and has minimal foreign body response with accelerated
angiogenesis.12 To date, CS has been used in the medical field as wound dressings,13
space filling implants14 and drug delivery systems.13a, 15 This work focuses on the
development of a delivery platform for the stable presentation of curcumin that preserves
the major pharmacologically relevant functional groups of curcumin for post-release
efficacy.
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Materials and Methods
Materials. Chitosan 90/200 was purchased from Heppe Medical Chitosan, GmbH (Halle,
Germany). Curcumin (85% pure; demethoxycurcumin and bis-demethoxycurcumin as
impurities), acetic acid, ethyl acetate (HPLC grade), 1-octanol, ascorbic acid, 2,2’-
diphenylpicrylhydrazyl (DPPH), dichloromethane, glutaric anhydride, methanol (HPLC
grade), 4-dimethylaminopyridine (DMAP), triethylamine, N-hydroxysulfosuccinimide
(sulfo-NHS), 1-Ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride (EDC),
diethylether, polyethylene glycol sorbitan monolaurate (Tween 20), butylated hydroxyl
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toluene (BHT), Folin Ciocalteu reagent and 10x phosphate-buffered saline were
purchased from Sigma Aldrich (St. Louis, MO, USA) and used as received unless
otherwise noted. Magnesium sulfate was purchased from Fisher Scientific (Pittsburgh,
PA USA). Dextran molecular weight standards were purchased from PolySciences, Inc
(Warrington, PA).
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Methods. Fourier transform mass spectrometry, ESI-MS, was performed on a MS-
FTICR-MSn, LTQ FT (Thermo Electron Corp., Waltham, MA). UV-visible spectroscopy
was performed on a Cary 100 spectrometer (Agilent Technologies, Santa Clara, CA) and
fluorescence spectroscopy on a Cary Eclipse Fluorimeter (Agilent Technologies, Santa
Clara, CA). Gel Permeation Chromatography was performed on a Waters 1215 binary
pump and 2420 evaporative light scattering detector (Waters Corporation, Milford MA).
Zeta potential measurements were made on a Brookhaven Zeta PALS analyzer
(Brookhaven Instruments Corp., Holtsville, NY). SEM imaging was conducted on a Zeiss
Supra 35 (Carl Zeiss AG, Jena, Germany). SEM samples were sputter coated with a
gold/palladium alloy prior to imaging.
Synthesis of 5-(4-((1E, 6E)-7-(4-hydroxy-3-methoxyphenyl)-3,5-dioxohepta-1,6-
dienyl)-2-methoxyphenoxy)-5-oxopentanoic acid (acid-curcumin). Acid-curcumin
was synthesized via a modification of published protocols.16 The reaction was performed
under air-free conditions using standard Schlenk reaction techniques. Tetrahydrofuran
(THF) was dried in a Pure-solv solvent purification system (Innovative Technology, Inc.,
Amesbury, MA). Curcumin (2.7 mmol) and DMAP (0.5 mmol) were dissolved in 100.0
mL dry THF. After stirring for 10 min., triethylamine (4.0 mmol) was added to the
reaction mixture. Glutaric anhydride (3.0 mmol) was dissolved in 5.0 mL THF and added
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drop-wise to the reaction mixture via addition funnel. A reflux condenser was attached to
the reaction vessel and the mixture refluxed overnight. Next, THF was removed using a
rotatory evaporator producing a dark red oil. The oil was collected with 55.0 mL ethyl
acetate and washed with 15.0 mL 1 M HCl. The organic layer was set aside and the
aqueous layer was washed with 50.0 mL ethyl acetate, three times. The combined organic
layers were dried over MgSO4 and gravity filtered. Solvent was removed on a rotatory
evaporator. 50 mg of the crude product was dissolved in 10.0 mL dichloromethane and
loaded onto a silica gel column. Elution with dichloromethane:methanol (95:5) resulted
in isolation of the title compound as an orange solid.
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Synthesis of curcumin-modified glucosamine. Acid-curcumin (21 ꢀmol) was
dissolved in 500 ꢀL methanol and added to 5.0 mL PBS buffer. Sulfo-NHS (23 ꢀmol)
was added to the acid-curcumin mixture and allowed to stir for 5 min at room
temperature. The mixture was then added to D-glucosamine-HCl (21 ꢀmol) dissolved in
15.0 mL 1% acetic acid. EDC (23 ꢀmol) was added and the reaction stirred for 24 hrs in
the dark at room temperature. The product was then extracted in 20.0 mL
dichloromethane, three times, then dried over MgSO4, filtered and dried on a rotatory
evaporator. The product was purified with column chromatography (silica gel; 95:5 –
50:50 DCM: methanol gradient). ESI(+)-MS: calcd. for C32H33O13N: 639.2. Found: 639.3
[M - H]+. Percent yield: 18.4%.
Synthesis of curcumin-chitosan polymer. Acid-curcumin (7.2 ꢀmol) was dissolved in
5.0 mL PBS buffer with 200 µL of methanol. Sulfo-NHS (7.9 ꢀmol) was added directly
to the solution and allowed to stir at room temperature for 5 min. The solution was then
added slowly to a solution of chitosan 90/200 (100 mg, 90 % deacetylated, 151-350 mPas
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viscosity, Mw 200-300 kDa) dissolved in 15.0 mL 1% acetic acid. EDC (7.9 ꢀmol) was
added to the mixture and stirred for 24 hrs in the dark. The solution was dialyzed for 72
hrs (MWCO = 3400 Daltons) in the dark followed by lyophilization to obtain a yellow
fibrous polymer. The product was then washed repeatedly with 70% ethanol until no
trace of curcumin was detected in the rinse (monitored spectroscopically at 420 nm).
Determination of yield of curcumin-chitosan polymer. Curcumin-chitosan polymer
was synthesized as above, with the exception that following dialysis the crude mixture
was extracted with ethyl acetate until no absorbance at 415 nm was noted via UV-vis
spectroscopy in the extracts. The total acid-curcumin in the extracts was then determined
from a calibration curve of acid-curcumin in ethyl acetate.
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Gel permeation chromatography of chitosan and curcumin-chitosan polymer. 0.2 %
w/v Chitosan and curcumin-chitosan polymer samples were stirred overnight in 0.3 M
acetic acid/0.1 M ammonium acetate, then filtered through a 0.22 um syringe filter. 100
uL samples were injected into an UltraHydrogel Linear column (7.8 mm I.D. x 30 cm,
Waters) and eluted with 0.3 M acetic acid/0.1 M ammonium acetate at 0.5 mL/min flow
rate. Average number molecular weights were calculated using a calibration of Dextran
standards weighing 43,500, 66,700, 124,000, 196,000, 277,000, and 401,000 Da.
Determination of curcumin-chitosan polymer isoelectric point. The isoelectric point
of the polymer was determined using a ZetaPALS Zeta Potential Analyzer with a BI-
ZTU Titration Unit (Brookhaven Instruments, Holtsville, NY) auto-pH titrator. pH values
were established via titration with nitric acid and potassium hydroxide.
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Curcumin stability in solution as hybrid polymer. Curcumin-chitosan polymer (100
mg) was dissolved in 100.0 mL DI H2O. The solution absorbance at 430 nm was
measured once a week for 1 month as a 1:10 dilution.
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Curcumin release studies. Carbonic anhydrase esterase activity was assayed against p-
nitrophenyl acetate using the method of Polat, et al.17 Carbonic anhydrase esterase
activity, as measured against p-nitrophenyl acetate, was determined to be 1.7
0.3
U/min/mL. One unit of enzyme activity was based on release of 1 ꢀmol p-nitrophenol per
minute at room temperature. Curcumin release with and without enzyme were conducted
in a release solution consisting of 0.05 M trizma buffer with 0.025 M sulfuric acid (pH
7.0) prepared in nanopure water and 0.057 M L-ascorbic acid as preservative. Curcumin-
chitosan polymer (5 mg) was added to 2.9 ml of release solution. At the same time, 0.125
mg/mL carbonic anhydrase buffer was prepared in the L-ascorbic acid buffer. To initiate
the enzyme-mediated release study, 2.9 mL of the L-ascorbic acid buffer and 0.1 mL of
the carbonic anhydrase buffer were placed into a centrifuge tube. As a control, the release
study was performed in the above manner with the omission of carbonic anhydrase.
Release study solutions were then covered in aluminum foil and placed in the dark at
37°C for release measurements.
For each time point measurement, the sample tubes were centrifuged at 2500 rpm for 5
minutes and supernatants were transferred to new centrifuge tubes. The curcumin-
chitosan polymer pellets were re-suspended in 3.0 mL fresh release buffer. 3.0 ml of
0.1% (w/v) BHT (as preservative) solution in ethyl acetate was added to each
supernatant. The supernatant solutions were mixed vigorously and the solvent layers were
allowed to separate for five minutes. Curcumin released into solution was quantified by
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measuring absorbance in the ethyl acetate layer at 420nm in a quartz cuvette. The process
was repeated over a period of nineteen days.
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Released curcumin sample preparation for DPPH and total phenolic content assays.
100.0 mg of curcumin-chitosan polymer was dissolved in 5.0 ml of 1% acetic acid and
wrapped aluminum foil to protect against photo-degradation. After 5 minutes, 5.0 ml of
ethyl acetate was added to the acetic acid solution, which was then vortexed. The
yellowish layer of ethyl acetate was carefully separated using a micropipette and
evaporated via rotavap. The resulting yellowish solid was re-dissolved in 4.0 ml methanol
and UV-Vis absorbance was measured at 420 nm. The average concentration of three
trials was calculated using curcumin’s standard curve. For total phenolic content
determination, the extracted yellow solid obtained via rotavap was re-dissolved in 2.0 ml
methanol and UV-Vis absorbance was measured at 420 nm. The average concentration of
three trials was calculated using the former equation for standard curve of curcumin in
methanol.
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Antioxidant studies. Determination of antioxidant capacity using DPPH was derived
from published protocols.18 Due to solubility differences between acid-curcumin and
curcumin-chitosan polymer, the DPPH studies were performed under two sets of solvent
conditions.
Curcumin/acid-curcumin/curcumin-glucosamine conjugate DPPH studies: 50 ꢀL of
various concentrations (0.05–0.2 mg/mL) of curcumin, acid-curcumin, or curcumin-
glucosamine conjugate were added to 5.0 mL 0.004% (DPPH) in methanol. The solution
was incubated in the dark for 30 min then the absorbance was read at 517 nm.
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Curcumin/curcumin-chitosan polymer/chitosan DPPH studies: Various concentrations
(0.05–0.2 mg/mL) of curcumin, curcumin-chitosan polymer, chitosan, and released
curcumin were prepared in 5.0 mL 1% acetic acid containing 0.025% Tween 20.
Curcumin equivalents to the curcumin-chitosan polymer were determined from a
calibration curve of curcumin in 1% acetic acid containing 0.025% Tween 20. To these
solutions with curcumin, chitosan, or curcumin-chitosan polymer, 1.0 mL of 0.004%
DPPH in methanol was added. The solution was incubated in the dark at 37°C for 30
min. then the absorbance was read at 515 nm. For both DPPH studies, radical scavenging
percentages were calculated from:
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(Control OD – Sample OD)/Control OD) x 100
where OD = optical density.
Total phenolic content. The total phenolic content in released curcumin was determined
using Folin Ciocalteu (FC) reagent via a method developed by Singleton and Rossi
expressing total phenolic content as Gallic acid equivalents.19 A 0.5 ml solution of
curcumin in methanol (3.8 to 18.9) µg/ml was mixed with 2.5 ml of 10% (v/v) FC
reagent and 2.5 ml of 7.5% (w/v) Na2CO3 prepared in the deionized water. A blank
solution was simultaneously prepared with 0.5 ml methanol, 2.5 ml 10 % (v/v) FC
reagent and 2.5 ml of 7.5 % (w/v) Na2CO3 prepared in the deionized water. The samples
were incubated in a thermostat at 37° C for 120 minutes followed by measuring the
absorbance at 765 nm. This procedure is repeated for standard curcumin (20 to 100)
ug/ml and Gallic acid (20 to 100) µg/ml respectively. The phenolic contents of the
extracted curcumin and standard curcumin are expressed as the Gallic acid equivalent
(mg of GA/g of extract) using a standard curve of phenolic content of Gallic acid from
the assay.
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Results and Discussion
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Curcumin modification. Curcumin has been modified with a pendant carboxylic acid
via standard coupling chemistry with glutaric anhydride utilizing DMAP and
triethylamine, resulting in “acid-curcumin”, Scheme S1.16 Isolation of the mono-
carboxylate derivative was achieved through column chromatography. This modification
allows facile conjugation of curcumin to amine bearing molecules such as chitosan and
D-glucosamine. High resolution Fourier transform mass spectrometry conclusively
identifies the identity of the product with a parent ion peak found at 483.16 amu [M+H]+.
Characterization of acid-curcumin via UV-visible spectroscopy and fluorescence
measurements was undertaken to verify that the spectral properties of curcumin were
minimally affected by the modification procedure (Figure S1). The ability to retain the
optical properties of curcumin throughout the conjugation process is deemed critical to
retaining the therapeutic properties of curcumin, as breakdown of the curcumin backbone
to less therapeutically relevant compounds is accompanied by a loss in the signature
absorbance/fluorescence bands for curcumin.20 The UV-visible spectra of curcumin and
acid-curcumin in DMSO are nearly identical, with only a 2 nm blue shift from 418 to 416
nm upon modification of curcumin, suggesting that the chromophore backbone of
curcumin is unperturbed by the conjugation reaction, Figure S1(A). Likewise,
fluorescence emission spectra of curcumin and acid-modified curcumin reveal only a 5
nm blue shift for the product from 518 to 513 nm, Figure S1(B). Comparison of the
infrared spectra of curcumin vs. modified curcumin reveals that the compounds have
nearly identical IR spectra with two important exceptions, Figure S2. The IR spectrum
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for acid-modified curcumin displays -C=O stretching bands at 1705 and 1755 cm-1,
implying the addition of a carboxylic acid to the curcumin structure, and the stretching
band related to the phenolic –OH of curcumin at 3504 cm-1 has been attenuated in the
product. The reduction in intensity for the phenolic -OH band implies that the
modification of one phenol moiety through esterification was successful.
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Conjugation of acid-curcumin to D-glucosamine. Addition of a pendant carboxylic
moiety to curcumin allows for facile conjugation to the amine moiety of chitosan.
However, as a feasibility test for the curcumin-chitosan conjugation, acid-modified
curcumin was conjugated to D-glucosamine using standard amide bond forming
Scheme 1.
chemistry, Scheme 1. Reacting a 1 to 1 molar ratio of acid-modified curcumin with D-
glucosamine in the presence of a small excess of sulfo-NHS and EDC affords the
curcumin glucosamine conjugate. The conjugation reaction proceeds with a yield of
18.4%. Curcumin-glucosamine conjugation has been confirmed by mass spectrometry
and FTIR. The main evidence of the successful conjugation is found in FT-MS analysis,
which reveals the expected peak for the conjugate at 639.3 [M-H]+, Figure 2. The
infrared spectrum of the curcumin-glucosamine conjugate contains strong stretching
bands for both glucosamine and curcumin as well as a strong carbonyl stretching band at
1763 cm-1, Figure S3.
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Conjugation of acid-modified curcumin to glucosamine minimally affects the
spectroscopic properties of the curcumin chromophore, as evidenced in the optical
spectra shown in Figure S4. The UV-visible absorption maximum and fluorescence
emission maximum at 420 and 539 nm, respectively, are within 5 nm of those reported
for curcumin.
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Figure 2. FT-MS (positive ion mode) of the curcumin-glucosamine conjugate.
Conjugation of acid-curcumin to chitosan. The successful conjugation reaction
between curcumin and glucosamine was used in the analogous reaction between acid-
curcumin and chitosan, Scheme 2. Reacting acid-curcumin with chitosan in the presence
Scheme 2.
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of sulfo-NHS and EDC followed by dialysis and lyophilization yielded a spongy
polymeric substance that bears the morphological characteristics of chitosan and the
optical properties of curcumin, Figure 3. Due to the low solubility of curcumin in 1%
acetic acid (even with the assistance of methanol) and the limitations of dissolving
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(A)
(B)
Figure 3. A) SEM image of the curcumin-chitosan hybrid polymer displaying a fibrous
morphology. EHT = 2kV (B) Photograph of the curcumin-chitosan hybrid polymer
reveals a bright orange-yellow color reminiscent of curcumin and a spongy consistency
characteristic of chitosan.
chitosan without the solution becoming overly viscous, it was practical to use an acid-
curcumin-chitosan ratio equivalent to labeling chitosan on 1% of the available amine
groups. Extraction of the crude polymer with ethyl acetate leads to recovery of 32.2% of
the polymer, suggesting to a yield of 67.7% for the conjugation reaction. For a polymer
molecular weight of 200-300 kDa and 90% deacetylation, this is equivalent to the
addition of 600-900 molecules of curcumin per polymer strand. SEM images of the
polymer reveal a fibrous polymeric structure that is typical for chitosan, Figure 3(A).21
The curcumin-chitosan polymer has a deep orange-yellow color that is typical of
curcumin, Figure 3(B). The polymer, as an acetate salt is soluble in 18 Mꢁ water in
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which it acts as a weak acid. As much as 5 mg/mL of the polymer can be dissolved in
plain water. The curcumin-chitsosan polymer can also be dissolved in dilute HCl and
then successfully dispersed in PBS buffer or cell culture media up to 4 mg/mL
concentration. Gel permeation chromatography (GPC), Figure S5, reveals that the
polymer has an increased apparent number average molecular weight (Mn) after
conjugation. Calibration with dextran standards yields Mn for chitosan as 299517 Da and
the curcumin-chitosan polymer as 322,822 Da. This gain in mass overestimates the
degree of curcumin addition at conjugation to 4.9% of available amines on chitosan.
Theoretically, a 100% yield would result in only 1% labelling. This suggests that
conjugation of curcumin to chitosan either alters the shape of the polymer in solution or
the rigidity of the polymer, thus artificially skewing the apparent Mn measurement with
GPC.22
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The FTIR spectrum of the curcumin-chitosan polymer contains all of the peaks that are
present in the curcumin-glucosamine conjugate, Figure S3. Both samples contain an
amide carbonyl stretching band at 1763 cm-1 indicating a successful coupling. In the
polymer, however, the mass ratio of curcumin to chitosan is low (on the order of 1%) and
so the IR bands associated with curcumin are weaker than in the curcumin-glucosamine
spectrum.
The conjugation of acid-curcumin to CS was also monitored by measuring the zeta
potential of the polymer before and after modification. As the amine groups on CS are pH
labile, the conjugation of curcumin to those amine groups and subsequent conversion to
uncharged amide groups will affect the overall charge on the polymer at a given pH. A
plot of zeta potential vs. pH for both chitosan and the curcumin-chitosan polymer is
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shown in Figure S6. It is evident from the shift in isoelectric point to higher pH (7.4 to
8.1) for the polymer that the conjugation reaction has reduced the number of free charge-
bearing amines on chitosan.
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Analogous to the curcumin-glucosamine conjugation, the strong optical absorption
profile of curcumin remains intact after conjugation to chitosan. However, the absorbance
maximum for curcumin
has blue-shifted to 380
nm (vs. 420 nm for
curcumin), Figure 4.
This may reflect the
fact that the curcumin-
Figure 4. UV/visible absorption spectrum (solid line) and
chitosan spectrum was
fluorescence emission spectrum (dashed line) of curcumin-
collected in acidic
chitosan hybrid polymer in 1% acetic acid.
aqueous
solution,
whereas aqueous spectra for unconjugated curcumin are collected in basic aqueous or
organic media.23 The fluorescence emission of curcumin remains after conjugation to
chitosan, with an emission maximum of 538 nm in 1% acetic acid, Figure 4.
Previous reports have demonstrated that curcumin is 90% decomposed within 30
minutes in aqueous environments.20 However, the curcumin-chitosan polymer was stable
in solution in DI H2O for up to one month with minimal change in solubility and optical
(absorbance and fluorescence) properties, confirming reports of the protective effect
imparted on curcumin by chitosan.24 The ability of polymer conjugation to prolong
curcumin solution lifetime and increase aqueous solubility has also been reported when
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using poly(ethylene glycol) (PEG) conjugates, where curcumin was detected in solution
up to 24 hours after exposure to cells.25
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Antioxidant capacity of curcumin and curcumin-derivatives. In order to test the
antioxidant capacity of curcumin after modification, a DPPH radical scavenging assay
was performed on curcumin, acid-curcumin, curcumin-glucosamine conjugate and the
curcumin-chitosan polymer.18a DPPH assays for the curcumin-glucosamine conjugate
were performed in methanol, however the poor solubility of the curcumin-chitosan
polymer was prohibitive to performing the DPPH assay on the polymeric form using the
same conditions as the other compounds, and required a modified form of the assay using
1% acetic acid with 0.025% Tween
100.0
20. For each solvent condition, the
80.0
radical scavenging abilities for the
60.0
various reagents were compared to
that of unmodified curcumin,
Figure 5. Before modification,
curcumin displayed 93% radical
scavenging activity. Modification
of one of the phenol groups of
curcumin lowers the antioxidant
capacity to 85%. The reduced
40.0
20.0
0.0
0.1
0.2
0.3
0.5
1
Chemical Equivalents
Figure 5. Radical scavenging assay results for
curcumin (white bar), acid-modified curcumin
(Striped bar), and curcumin-glucosamine
conjugate (black bar) in methanol.
antioxidant capacity of curcumin-glucosamine is most likely due to usage of one of the
phenol moieties on curcumin for tethering the molecule. Conjugation of curcumin to
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glucosamine reduces radical scavenging capabilities to 60%, Figure 5. The further
reduction in antioxidant capacity may reflect the presence of hydrogen bonding
interactions between glucosamine and curcumin that stabilize groups that would typically
participate in radical quenching reactions. However, the tethered curcumin molecules
display 60% activity, rather than 50%, of the radical quenching ability of curcumin which
may reflect antioxidant activity of the β-diketone moiety contributing to radical
quenching reactions.3c
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The antioxidant capacity of curcumin-chitosan polymer, chitosan and post-release
curcumin (vide infra) were also assessed using a DPPH assay, but was performed in 1%
acetic acid with 0.025% Tween 20 due to the limited solubility of the polymer in
methanol. Tween 20 was included to effectively solubilize curcumin in the aqueous
solution for use as a control for the DPPH assay. Similar to the DPPH assay in methanol,
curcumin was found to demonstrate 92.2% radical quenching at a 1:1 ratio with DPPH.
Curcumin-chitosan polymer performed in a manner analogous to the curcumin-
glucosamine conjugate with radical quenching ability reduced to 53.2% after
modification and conjugation of
100
curcumin, Figure 6. The reduction
in radical quenching ability is
again contributed to usage of one
of the two phenol groups of
curcumin for the conjugation
reaction. As a control, the radical
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80
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Chemical Equivalents
Figure 6. Radical scavenging assay results for
curcumin (white bar), curcumin-chitosan
polymer (striped bar), and released-curcumin
(black bar) in 1% acetic acid.
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chitosan was also measured and found to be 12% (data not shown). This may explain
why curcumin-chitosan polymer displays radical scavenging above 50% even though half
of the available phenol groups have been modified.
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Notably, the curcumin released from the curcumin-chitosan polymer (vide infra) has
regained all of its radical scavenging
capabilities.
This
implies
that
Table 1. Phenolic content assay results for
covalently bound curcumin is released
through ester hydrolysis, which would
result in re-formation of the phenol
that was modified to attach curcumin
to the polymer. The reconstitution of
curcumin further confirmed in two
ways. First, ESI-MS confirmed the
presence of curcumin as the dominant
unmodified and post-release curcumin.
Sample
Total phenolic
content (mg GAE/g)
Extracted curcumin
Standard curcumin
422.9 0.7
372.5 0.9
Standard
(values taken from ref.
curcumin 448.4 8.3
19, 26
)
peak (M+H 369.13). In order to confirm that this peak was not the result of fragmentation
during ionization in the mass spectrometer, phenolic content assays were performed on
the extracted curcumin following release from the polymer, Table 1. The assay, which
determines the total amount of phenol groups in a sample relative to a Gallic acid
standard, confirms that the post-released curcumin has a phenolic assay count very
similar to the unmodified curcumin used in this study.19
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Curcumin release studies. Release studies were performed on the curcumin-chitosan
polymer in its fibrous polymer morphology both with and without the presence of
carbonic anhydrase. Carbonic anhydrase was chosen for its well-characterized ability to
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cleave phenolic esters such
120
as the one used to tether
100
80
curcumin to the chitosan
60
backbone.
curcumin-chitosan
polymer, curcumin
For
the
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is
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20
Time (Days)
released steadily over the
course of 19 days from the
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order kinetics, Figure 7.27
The effective dose of
Figure 7. Curcumin release from curcumin-chitosan
polymer with (black squares) and without (white
diamonds) carbonic anhydrase.
curcumin released over this time is 230 120 nM curcumin/mg polymer/mL solution
based on 1% curcumin loading on the polymer. The release rates measured here are
dramatically longer than previous reports involving PEG-conjugated curcumin
derivatives, suggesting that the curcumin-chitosan polymer may be more useful in
applications where a longer release time is needed.25 Curcumin is released from the
polymer at the same rate regardless of the presence of functionally competent carbonic
anhydrase; suggesting that the length of the connecting tether between chitosan and
curcumin may not be sufficient for allowing carbonic anhydrase to cleave the ester bond,
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or that simple ester hydrolysis is likely to be the main release pathway for the
curcumin.
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Conclusions
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Curcumin is a potent antioxidant and is a promising agent in treatments and therapies
against diseases and injuries, such as cancer and inflammation; however, its low
solubility limits its bioavailability and clinical efficacy. To overcome these limitations,
curcumin has been conjugated to chitosan polymer. Additionally, curcumin was
conjugated to the small molecule analog of chitosan, glucosamine, to further characterize
the conjugation product. Covalent conjugation of curcumin to chitosan not only protects
curcumin from rapid degradation, but also provides a means for controlled delivery to
various tissues for specific applications. The attachment modality of curcumin to chitosan
reported here contains biodegradable linkages to facilitate both hydrolytic and enzyme
catalyzed release and was designed to release curcumin in its pre-modification form.
Non-enzyme catalyzed hydrolytic release was determined to be the predominant release
mechanism in this system. The antioxidant capacity of curcumin throughout the
conjugation and release processes has been monitored. Release studies have been
performed to compare the rates of enzymatic vs. non-enzymatic release. Though
antioxidant capacity is temporarily reduced, due to modification of one of the phenol
groups, it is restored once curcumin is released from the polymer. The conjugation to
chitosan therefor provides a viable strategy to stabilize curcumin for applications
involving long-term release delivery. The hydrolysis-triggered release of curcumin from
the polymer provides a steady concentration of curcumin over the period of the release
study. This indicates that curcumin-chitosan polymer is a promising candidate carrier to
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deliver therapeutic doses of curcumin over extended periods of time while maintaining
potency for antioxidant applications.
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ASSOCIATED CONTENT
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Supporting Information. Additional spectra for modified-curcumin and Zeta potential
measurements on the curcumin-chitosan polymer are located in the supporting
information. This material is available free of charge via the Internet at
http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
* Email: martin.otoole@louisville.edu
Department of Bioengingeering
University of Louisville
Lutz Hall Rm 411
Author Contributions
The manuscript was written through contributions of all authors. All authors have given
approval to the final version of the manuscript. ‡These authors contributed equally.
Funding Sources
Support for this work was in part provided by NASA Grants NNX10AJ36G and
NNX13AD33A and the University of Louisville department of Bioengineering.
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ACKNOWLEDGMENT
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The authors wish to acknowledge Joe Williams and Dhruvinkumar Patel for their
assistance with SEM imaging and Patrick Hoblitzell for his contributions to discussions
about the project.
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