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Dhanavel 2017
Dhanavel 2017
PII: S0928-4931(16)31449-7
DOI: doi: 10.1016/j.msec.2017.03.058
Reference: MSC 7566
To appear in: Materials Science & Engineering C
Received date: 23 September 2016
Revised date: 4 January 2017
Accepted date: 3 March 2017
Please cite this article as: S Dhanavel, E A K Nivethaa, V Narayanan, A Stephen , In vitro
cytotoxicity study of dual drug loaded chitosan/palladium nanocomposite towards HT-29
cancer cells. The address for the corresponding author was captured as affiliation for all
authors. Please check if appropriate. Msc(2016), doi: 10.1016/j.msec.2017.03.058
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*E-mail: stephen_arum@hotmail.com
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Phone: 044-22202802, Fax. 044-22351269
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Abstract
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Conjugated drug delivery has gained immense interest due to the possibility of overcoming the
resistance of cancer cells to a specific drug when treated using it for a period of time. In the
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present study, CS/Pd nanocomposite has been prepared using cost effective chemical reduction
method and has been used for the delivery of curcumin (CUR) and 5-Fluorouracil (5-FU)
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separately and in a conjugated form. The prepared nanocomposite before and after drug
encapsulation have been studied using various characterization techniques. The release of drugs
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from the nanocomposite with respect to time has been analyzed and the release kinetics has also
been studied. The release profile is mostly seen to adhere to zero order kinetics which represents
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the constant release of drugs from drug delivery system. This is the most favored release kinetic
as this leads to the prolonged release of the drug, thus leading to a reduction in the number of
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doses administered. The cytotoxicity of the drug loaded nanocomposites on colon cancer cells
has been studied, which shows the effectiveness of the composite system towards successfully
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1. Introduction
In recent years, cancer is one of the most lethal diseases and it continues to be a global burden. It
is responsible for the death of more than 8 million people worldwide each year. Death
occurrence due to cancer has been estimated to be 13.1 million in 2030 [1-3]. Among them,
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colon cancer is the third most common malignant tumor, accounting for 1.4 million deaths each
year [4, 5]. Currently, several approaches, such as surgery, photodynamic therapy, photothermal
therapy, radiotherapy and chemotherapy have been applied for the treatment of colon cancer.
Among the various techniques, chemotherapy is the most effective and inexpensive method [6,
7]. However, the application of a single drug chemotherapy is still limited by many barriers such
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as side effects caused by high or repeated drug dosing, poor bioavailability, fast renal clearance
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and multi drug resistance [8]. To overcome these barriers, recently combination chemotherapy of
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multiple anticancer drugs has been developed. It is able to reduce the drug resistance as a result
of lower dose of each drug. It has been reported that the use of multiple drugs influences
imaging, energy devices and targeted drug delivery [11-13]. The large surface to volume ratio of
nanoparticles and their size, optoelectronic properties, ability to carry other compounds, ability
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to bind and their adsorption properties make them suitable for biomedical applications. Apart
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from these, nanoparticles can improve the bioavailability, protect drug from degradation and also
control the release rate (i.e.) it can enable the prolonged release of drug. Hence, these unique
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characteristics of nanoparticles offer a platform for their use as an effective drug delivery system
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[14]. Among the various nanoparticles, noble metal nanoparticles have attracted more attention
because of their reduced toxicity, unique optical, chemical and electronic properties when
compared to other materials [15]. Especially, palladium nanoparticles have gained attention in
recent years, for use in various applications such as catalyst, biosensor for the detection of
analytes, as antimicrobial agent and in dental appliances [16-18]. Recently, Anaelle Dumas et al.
suggested that low toxicity of palladium nanoparticles qualify them as a future key players in the
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nanomedical field [19]. Andrea Schmidt et al. has reported the use of palladium cages for the
cisplatin delivery and have also studied its cytotoxic effect on human cancer cells. They have
also reported that cages encapsulating cisplatin showed improved cytotoxic effect against human
ovarian cancer cells compared to free cisplatin [20]. Seed-mediated synthesis of palladium-gold
(Pd-Au) bimetallic nanostructures for the photothermal cancer therapy has been investigated by
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Andrew J. McGrath et al [21]. This assures that, palladium nanoparticles can play a vital role in
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eliminating cancer cells. However, the major drawback of utilizing Pd nanoparticles as drug
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delivery carrier is its agglomeration while reducing from its precursors. Also the formation of
palladium with larger particle size hinders its usage in medical applications. To overcome these
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problems, palladium nanoparticles are generally dispersed on polymer or inorganic supports such
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as MCM-41( Mobil Composition of Matter No. 41) and SBA-15 (Santa Barbara Amorphous-15)
which helps in preventing agglomeration and introducing homogeneity [22, 23]. Recently,
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attempts have been made using biopolymer based stabilizing agent to immobilize or stabilize
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distributed (β-1→4) linked d-glucosamine and N-acetyl-d-glucosamine units. Among the various
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biopolymers, chitosan has received much attention because of its outstanding biological and
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toxicity, unique muco-adhesive and low-immunogenicity make it the most suitable candidate for
various applications such as, biopolymer batteries, sensors and biomedical applications [25, 26].
Majorly, presence of chelating sites (NH2 and OH) in its functionality aids in chemical reactions
as metal ion sorption through electrostatic attraction and ion exchange for metal anions in acidic
solutions [27]. There are a number of reports available on chitosan based framework for drug
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delivery applications [28, 29]. In our previous report, we have shown the preparation of chitosan
stabilized noble metal NPs, and have used them for the delivery of 5-FU [26, 29, 30]. Similar
mechanisms can be expected for Pd NPs immobilized chitosan for the cancer therapy.
The current treatments for colon cancer such as chemotherapy are mostly based on
5-Fluorouracil (5-FU). It inhibits the growth of cancer cells by interfering with DNA. The major
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drawback is that, frequent exposure to 5-FU can lead to acquired drug resistance (ADR) which
will cause severe side effects [31, 32]. Therefore a new strategy is needed to deliver high
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concentration of 5-FU intra-cellularly to the cancer cells to overcome ADR. Therefore, curcumin
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is chosen as another drug to improve the efficacy of 5-FU.
Curcumin (CUR), a hydrophobic polyphenol derived from turmeric plant, has been used for a
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long time in Indian medicine for the treatment of several diseases. It has various therapeutic
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properties [33]. Recently, CUR based combinatory therapy has been explored due to its unique
features, such as suppression of drug resistance through sensitization of cancer cells, restricting
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the cellular cell invasiveness associated with the progression of cell growth. It is also well
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tolerated at high doses which is safe for humans [34]. Unfortunately, CUR as a cancer
therapeutic agent in cancer treatment has been limited due to its poor bioavailability and poor
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solubility [35]. A. Anitha et al. has investigated the effectiveness of 5-fluorouracil and curcumin
cancer cells [36]. To the best of our knowledge, the application of chitosan supported Pd NPs as
drug carrier for the cancer treatment has not yet been reported. Based on the previous reports,
improve the anticancer activity. In particular, this report focuses on the co-delivery of drugs and
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its effectiveness in cancer treatment to validate its feasibility in the combinatory therapy against
2.1. Materials
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Palladium (II) acetate (98%), chitosan (low molecular weight and ~85% deacetylated),
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Curcumin, 5-Fluorouracil with ≥ 99% and Dimethyl sulfoxide (DMSO) with 99% purity were
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purchased from Sigma Aldrich. Sodium tripolyphosphate with 98% purity and Tween 80 ultra-
pure was procured from Alfa Aesar. Sodium borohydride (99%) was obtained from Merck India.
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Solvents and all other chemicals used were of analytical grade. Double distilled water was used
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for the synthesis.
The X-ray diffraction (XRD) measurements were carried out at room temperature from GE
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X-ray diffraction system-XRD 3003 TT with CuKα1 radiation (λ=1.5406 Å) for 2θ = 5–70⁰ at a
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scan rate of 0.04°/Sec. The size and morphology of samples was done using HRTEM analysis
by Technai instrument operating at operating voltage of 200 kV. The surface morphology of the
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composite was examined using HITACHI SU-6600 FESEM. Elemental mapping was carried out
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using FEI Quanta-250 FEG. Fourier transform infrared spectra were obtained from Perkin-Elmer
FTIR system. Measurements were carried out by dispersing the sample in a KBr to analyzing the
peaks in the range 4000-450 cm-1. XPS measurements are performed with DAR400-XM 1000
(OMICRON Nanotechnologies, Germany) equipped with dual Al/Mg anodes as the X-ray
source. The Al anode was used to obtain the elemental and survey spectrum. All spectra were
calibrated using C 1s peak at 284.5 eV as a reference to exclude the charging effect on the
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sample. The UV-Vis studies and release profile was obtained using UV-Visible
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method [17]. Initially, chitosan stock solution at a concentration of 0.34% (w/v) was prepared by
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dissolving chitosan in 2% acetic acid. The prepared solution was filtered. 0.0329 M solution of
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palladium acetate was prepared by dissolving it in 2% acetic acid in order to get 5% (w/w)
palladium in the composite. The palladium solution was added drop wise into the chitosan
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solution which was kept under simultaneous stirring and sonication. 1.4mM of sodium
tripolyphosphate (TPP) solution was added to the above solution. Then, palladium nanoparticles
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immobilized chitosan was obtained by the in situ reduction of palladium acetate by the dropwise
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addition of 0.65 M sodium borohydride to the above solution. After stirring and sonication for 1
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h, the obtained precipitate was washed with double distilled water and dried in an air oven. In a
similar way, 10 and 15 weight percentage of palladium loaded chitosan matrix nanocomposite
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encapsulation for the synergistic therapy. In a typical synthesis procedure of 5-FU encapsulated
CS/Pd, 3.8mM 5-FU solution (dissolved in water) was added into the previously prepared
chitosan solution (dissolved in 2% acetic acid). The prepared solution was kept under
simultaneous stirring and sonication for 15 min. Tween 80 (0.5% v/v) and 1.4mM of Sodium
tripolyphosphate (TPP) solution was added to the prepared 5-FU containing solution. The ratio
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of the amount of chitosan to TPP was taken as 2:1 (v/v). Then the obtained suspension was
maintained under stirring and sonication for 2 h. Then Palladium acetate solution was added to
get 5% (w/w) palladium in that prepared suspension. 0.7M sodium borohydride was added to
reduce the palladium acetate into palladium. The product was decanted and centrifuged. Finally
the product was collected by freeze drying and it was used for further analysis. For the synthesis
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of CUR encapsulated CS/Pd, 1.32mM CUR solution (dissolved in ethanol) was added to the
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chitosan solution. For the case of co-encapsulation, 5-FU and CUR (1:1 ratio) were added
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simultaneously and the same procedure was followed as explained in synthesis of encapsulation
of 5-FU.
(NCCS), Pune, India. The cells were cultured in Minimal essential media supplemented with
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CO2 at 37 °C.
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Fig. 1a shows the XRD pattern of Chitosan/Pd nanocomposites containing different weight
percentages of palladium. The XRD pattern for all the composites are similar and shows the
presence of both chitosan and palladium. Two peaks at 2θ of 10.6° and 21.1° are observed,
indicating the semicrystalline nature of chitosan. The XRD pattern of chitosan is given in
supplementary information (Fig. S1). The peaks appeared at 39.5° and 46.2° is indexed to (111)
and (222) planes of the face centered cubic structure of Pd. The obtained diffraction pattern is in
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good agreement with the JCPDS card No. 87–0637. It is observed that intensity of the palladium
peaks were increased while increasing the weight percentage of palladium as 5, 10 and 15%. Fig.
1b shows the XRD pattern of 5-FU, CUR and 5-FU+CUR loaded composites. All the conjugates
show the presence of both chitosan and palladium (111) peaks. Apart from these, CUR peaks
were observed in CUR@CS/Pd and peaks concurred well with the previous reports [4, 35].
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Similarly, intense diffraction peaks of 5-FU are observed in 5-FU@CS/Pd. The XRD peaks of
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5-FU matches well with the JCPDS No. 39-1860 [30]. Presence of both 5-FU and CUR peaks
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affirms the encapsulation of 5-FU and CUR to the composite. The XRD pattern of commercial
palladium in the range of 4000–450 cm-1. The wide peak located at ~ 3409 cm-1 is attributed to
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the interstitial water and hydroxyl group, δ(OH) which overlaps with the N-H stretching band of
chitosan [37]. The peak at ~1665 cm-1 indicates the presence of bending vibration of NH2 group.
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The band at ~1565 cm-1is assigned to the NH3+ group. It can be observed that splitting of NH2
and NH3+ group varies in the composites with different weight percentage of palladium. The
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occurrence of differences is due to the neutralization of protonated amine group on increasing the
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amount of palladium. The intensity of the NH2 peak increases with addition of increased amount
of palladium, this proposes the incorporation of palladium into the chitosan matrix. The
transmittance peaks which appear at ~1388 cm-1 and ~1319 cm-1 can be ascribed to the C-H
bending and C-N stretching modes respectively. The peak observed nearly at ~995 cm-1 is due to
the NH2 twisting mode. The peak at ~1226 cm-1 is due to the C-O-C stretching vibration. The
~794 cm-1 peak represents the C-H out of plane vibration. While increasing the percentage of
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palladium, new peaks are observed at lower wavenumbers nearly in the region of ~500 cm-1
indicating the loading of palladium into the composites [29, 30, 38]. In comparison with the IR
spectrum of chitosan, the peak shift confirms the interaction of chitosan with palladium by
chelation [30, 39, 40]. In CUR loaded CS/Pd nanocomposite (Fig.3b.), the peak shift is observed
at ~1653 cm-1 which corresponds to the N-H deformation and new peaks appearing at ~1075
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cm-1 corresponds to the keto group of the CUR which confirms the encapsulation of CUR into
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CS/Pd. Fig 3c shows IR spectrum the 5-FU encapsulated CS/Pd nanocomposite. After 5-FU
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loading into the composite the amine peak is shifted from 1665 cm-1 to 1656 cm-1 and NH3+ peak
from 1565 cm-1 to 1555 cm-1. The peak at 665 cm-1 indicates the presence of C-H out of plane
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vibration of CF=CH. The observed shifting and enhancement of peak intensity at ~ 1650 cm-1 is
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due to the intermolecular hydrogen bonding of carbonyl and a fluorine moiety of 5-FU with the
N-H group of chitosan. While loading both the drugs into the composite, intensity of the amine
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peak (1651 cm-1) has increased (Fig.3d.). Moreover, the CF=CH peak and keto group were
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shifted to the lower wavenumber, 663 and 1010 cm-1 respectively. It suggests that, keto group of
CUR and fluorine moiety of 5-FU interacted with the CS/Pd nanocomposite [35]. Thus, from this
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study, it is confirmed that all the materials used in the preparation of chitosan conjugates have
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The UV–Vis spectra of palladium (II) acetate and CS/Pd-5% are shown in Fig. 4. The UV-vis
spectrum of palladium (II) acetate exhibits a peak around 415 nm which refers to the existence of
Pd2+ ions. The reduction of palladium (II) ions into palladium (0) in the chitosan matrix was
monitored by UV-Vis spectra under the addition of NaBH4. UV-Vis spectrum of CS/Pd shows
complete conversion of Pd(II) to Pd(0) by the absence of the peak at 415 nm which corresponds
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to Pd(II) species [41]. The formation of palladium nanoparticles with the absence of peak (no
Simultaneously, the reduction of palladium (II) species was observed by monitoring the changes
in color. Initially, chitosan- palladium (II) acetate showed pale yellow colored solution. After the
addition of NaBH4, it exhibits color change from pale yellow to black which indicates the
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formation of palladium nanoparticles.
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Inset of Fig. 4 shows the UV-Vis spectra of 5-FU, CUR and combined drug encapsulated CS/Pd
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264 nm where as in the combined system it is observed at 267 nm. CUR loaded system shows
the characteristic peak of CUR at 438 nm and in the combined system shows absorbance at
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439 nm. The observed peak shift is due to the interaction between the drug molecules as well as
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Fig 5 shows typical FESEM images of dual drug encapsulated system taken at low (a) and high
magnification (b). The high magnification image clearly shows that particles (white) are
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embedded in chitosan matrix (Grey area). Elemental mapping of co-encapsulated CS/Pd is given
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in Fig 5c. It reveals that the composite consists of C, O, N, F and Pd. It confirms the presence of
5-FU in the composite. In Fig 5d, nitrogen and palladium map implies the chelation of
palladium with the amine group of chitosan. Nitrogen and fluorine map represents the interaction
between 5-FU and amine group of chitosan in co-encapsulated CS/Pd (Fig 5e).
Fig 6a-c shows the HRTEM micrographs of CS/Pd at various magnifications. It can be observed
that polymer matrix type nanocomposite is formed with palladium nanoparticles (Dark area) as
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filler phase and chitosan as the polymer matrix. Micrograph reveals that palladium nanoparticles
are uniformly dispersed in chitosan and the morphology is found to be homogeneous. The size of
the palladium nanoparticles are measured and found to be ~3-4 nm using ImageJ software.
According to this result, CS/Pd nanocarrier with smaller particle size, exhibits larger effective
area which is favorable for a better encapsulation efficiency. Further, the interplanar d-spacing
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value was calculated as 0.227 nm, using the lattice resolved image of spherical palladium
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nanoparticles. It matches to the (111) plane of typical Pd metal (JCPDS No. 87–0637) and lattice
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constant calculated to be a = b = c = 0.398 nm. Thus from this point, it agrees well to the XRD
result. An increase in particle size and agglomeration are observed for the case of drug loaded Pd
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nanoparticles (Fig. 6d). This may be attributed to the binding of the drug molecule to more than
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one Pd nanoparticle [30].
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The XPS spectra have been recorded to identify the surface composition and the existing
chemical states of that element. The curve fitting of the XPS peaks were performed using Fityk
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software after removing the shirley background. The binding energy of Carbon was taken as a
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reference for charge correction. Fig 7a shows the full scan XPS spectrum of CS/Pd
nanocomposite. The peaks in the full scan spectra revealed the presence of Carbon, oxygen,
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nitrogen and palladium. The C 1S spectra (Fig 7b) deconvoluted into two peaks at binding
energies, 284.6 eV and 288.3eV corresponding to the C-C/C-H and C-N/C=O environments
respectively [44, 45]. The N-1S spectrum (Fig. 7c) consists of two peaks at 399.5 eV and
401.1 eV represents the presence of NH2 and NH3+ respectively. The high binding energy
appeared at 401.1 eV is due to the positively charged nitrogen atom which is due to the
protonation of amine groups of chitosan on dissolving in 2% acetic acid. The shift in the peak
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position of NH2 and NH3+ group when compared to the pure chitosan suggests that free amino
group –NH2, may have interaction with Pd by chelation, whereas the protonated amino group -
NH3+ has a strong coulombic interaction with TPP ion as reported for CeIII–chitosan complex
and chitosan/gold nanocomposite [30, 44, 46]. The two peaks at binding energies 533.4 eV and
535.6 eV represents the deconvoluted O 1S spectrum (Fig. 7d). The atomic concentration
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percentage of OH group in the CS/Pd nanocomposite was decreased to 42% when compared to
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85% in the pure chitosan [30]. It may be owing to the chelation of palladium into the chitosan
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matrix as observed for the case of ethylene diamine functionalized carbon nanotubes and
chitosan iron complex [47-49]. It is well known that, OH and amine groups are playing as active
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sites in chitosan. But the peak position of OH is not shifted as observed in the case of N-1S
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spectrum. This indicates that protonated amine group in chitosan plays a major role for the
formation of chitosan/Pd composite through amino group nitrogen atom on chitosan can provide
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isolated pairs of electrons to metal ions. It can be supported by our previous report [30, 50]. Fig.
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4e illustrates the palladium 3d spectrum. It exhibits two peaks corresponding to the binding
energy of 336.1 eV and 341.3 eV, which can be assigned to the 3d5/2 and 3d3/2 of Pd0
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respectively. The obtained binding energy values shows shift with the original metallic
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palladium by 0.7 eV which may be attributed to the binding of palladium with chitosan and
effect of particle size [30, 51]. Taking together the O 1s, Pd 3d and N 1s results, binding of
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palladium with the chelating sites of chitosan is evident from XPS analysis.
Drug loading and entrapment efficiency of CS/Pd nanocomposite was calculated as follows
The encapsulation and drug loading efficiency was calculated from the quantity of 5-FU or CUR
encapsulated to CS/Pd which was determined using UV-Vis analysis technique by measuring the
absorbance percentage of the characteristic peak (265 for 5-FU and 435 nm for CUR). The
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quantity of unbound 5-FU and CUR present in the supernatant prior to and after adsorbing onto
CS/Pd was measured against the spectra of standard 5-FU/CUR solutions with a known
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concentration. The entrapment efficiency and loading efficiency of CUR from CUR loaded
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CS/Pd nanocomposite was estimated to be 96.33% and 18.19%. Similarly, for 5FU loaded
nanocomposite system, it was found to be 95.93% and 16.01% respectively. The obtained drug
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encapsulation and drug loading efficiency was listed in table 1. In the case of dual drug loaded
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system, entrapment & loading efficiency was determined as 94.09% & 11.66% for 5-FU and
92.65% & 16.39% for CUR. The estimated encapsulation efficiency of both the drugs in dual
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drug loaded system decreases when compared to the single drug loaded conjugates. It is
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depending on the number of drug and drug type used in the preparation. A similar behavior was
observed by Bo Xiao et al [4]. However, The obtained efficiency of 5-FU and CUR is higher
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than the CS/Gold -5FU nanoconjugates which was reported by Parvathy R. Chandran et al [52]
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A. Anitha et al [36].
In vitro drug release profile of 5-FU, CUR and dual (5-FU + CUR) drug loaded CS/Pd were
carried based on our reported protocol [26, 29, 30]. The studies were carried out in a phosphate
buffer saline (PBS) solution of ~pH 5 because of the well-known fact that cancerous cells have
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low pH when compared to the normal cells. Moreover, A. Anitha et al. reported that at lower pH,
enhanced release was observed from both 5-FU and CUR loaded thiolated chitosan nanoparticles
and they suggested that enhanced release in acidic pH was due to the sol-gel transition [28]. In
acidic condition, the polymer matrix swells due to protonation of primary amine group of
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20 mg of the nanocomposite (CS/Pd) synthesized with known amount of 5-FU was dispersed in
2 ml of prepared PBS solution. The prepared suspension was transferred into a dialysis bag
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(MWCO 1000 Da) and the ends of the dialysis bag were sealed and immersed into a 60 ml
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phosphate buffer solution which was kept under a constant slow magnetic stirring of 100 rpm at
37 °C for 72 hours. At specific time intervals, 3 ml of the solution was collected and
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simultaneously 3 mL of PBS was added in order to release the drug into a constant volume for a
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constant evaluation.
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The release rate and quantity of drug released from carriers is important prerequisite for the
5-FU [30] and CUR (given in supplementary information Fig. S5) were plotted to evaluate the
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quantity of drug. In brief, the known amount of drug is suspended in phosphate buffer saline and
serial dilution of this solution was done to obtain the remaining concentrations. The amount of
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5-FU released was then calculated using maximum wavelength of absorption at 265 nm against
the calibration curve. For the release of CUR, it was calculated at 435 nm against the respective
In vitro drug release profiles of 5-FU, CUR and dual (5-FU + CUR) drugs from nanoformulates
is shown in Fig 8a, b &c respectively. In order to determine the kinetics and release mechanisms,
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five different mathematical models were used to fit the release profiles of 5-FU and CUR from
CS/Pd nanocomposites.
K0t (3)
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First order model,
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1-exp (-K1t) (4)
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Korsmeyer-Peppas model
tn (5)
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Hixson-Crowell model
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Higuchi model
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KH ×t1/2 (7)
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Here, Mt/M∞ is % fractional drug release at time t. K0, K1, Kkp , Kc and KH are the zero order
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release constant, first order release constant, kosmeyer –Peppas constant, Hixson-Crowell release
constant and Higuchi release constant respectively. Zero order kinetics is highly suitable for the
long-term delivery systems. First order kinetics provides information regarding to the dissolution
process. Korsmeyer-Peppas model explains about the mechanism of drug release from devices of
different geometry through release exponent (n), when n < 0.5, it indicates Fickian diffusion
process (Case I transport), n = 0.5 represents Higuchi kinetics. For 0.5 < n > 0.85, the release
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profile represents the anomalous transport mechanism, which indicates the coupled effect of
diffusion and polymer relaxation/erosion process. When, n = 0.85 specifies the release from
emulsion was swelling-controlled, Case II transport mechanism. n > 0.85 indicates the Super
observed. Similarly, Hixson-Crowell kinetic describes the release from systems where there is a
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change in surface of drug carrier [54-56].
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The highest value of regression coefficient (R2) suggesting the good correlation between the drug
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release profile and the kinetic model. In other words regression coefficient is the indicator of the
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model prediction. The obtained correlation coefficients (R2) were summarized in Table.2 for the
CUR release from CS/Pd is shown in Fig 8a and the obtained release profile was fitted as three
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different regions, namely region I from 1 to 9 h, region II from 12 to 32 h and region III from 36
to 72 h, with various models. As in the case of CUR (Fig. 8a), initial burst release was observed
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as 34.7% and 30.48% for the release of curcumin from CUR@CS/Pd and dual drug loaded
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systems respectively. Normally, this burst release occurs only at the beginning which is due to
the diffusion of drug present at the surface of nanocomposite. In CUR loaded system, first region
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(1 to 9 h) fitted well with the Higuchi kinetics suggests that the drug release from a matrix
system which does not swell or degrade. The best fit for second region (12 to 32 h) was found to
be first order kinetics indicating the amount of drug released at each time was proportional to the
residual drug inside the drug carrier. The third region (36 to 72 h) concurred well with the case I
CUR release from dual delivery (Fig.8c) also exhibits three region (1to18, 22 to 39 & 44 to 72
h). First and second region follows the zero order kinetics which indicates the constant drug
release from a co-drug delivery system. The third region supported by the Korsmeyer-Peppas
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Initially, 28.31% of the drug was released from 5-FU loaded CS/Pd whereas in dual drug loaded
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system (Fig. 8c), 28.53% was released. In Fig 8b, Followed by the burst release, the first region
fitted well to the Higuchi kinetics (1 to 12 h) indicates that drug release follows a square root of
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time relationship. The second region fitted well to the Korsmeyer-Peppas model. Diffusional
constant (n) from the Korsmeyer-Peppas fitting indicates the drug release transport mechanism.
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The obtained value (n = 0.157) suggests that drug diffuses through and is released from the
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polymeric matrix with a Fickian diffusion mechanism (Case I transport). The Hixon-Crowell
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kinetics which fits well with third region suggests that drug release process is based on drug
erosion from drug carriers. The release of 5-FU obtained from co-delivery process differs from
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single delivery system (Fig. 8b). It is also having three regions (1 to 6, 9 to 44, 50 to 72 h).
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Similar to the CUR release, the first and second region fitted well to the zero order kinetics. It
was also observed that (third region), drug release follows Korsmeyer-Peppas which suggests
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that the release process from nanoparticles is controlled by anomalous, non-Fickian drug
diffusion. Non-Fickian drug diffusion attributed to the competing release through matrix
degradation.
From the analysis, CUR shows high burst release than 5-FU. But, followed by the burst effect,
slow release was observed when compared to the release of 5-FU. From the release data, it is
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seen that almost above 80% of the drug (5-FU) is released within 72 h which is higher compared
to the CUR release. The observed variation in the release rate of 5-FU and CUR is owing to the
differences in their hydrophobicity. Briefly, 5-FU is water soluble and it can readily diffuse into
hydrophilic-releasing media from the polymer matrix whereas CUR is hydrophobic which tends
to remain within polymer-inorganic network. So it exhibits a faster release compared to CUR [4,
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57]. However, the obtained variations in the co-delivery of drug from single delivery vehicle
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depends on the combinatory effect of erosion, swelling, polymer degradation and its pore
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diffusion [4]. To some extent, the slow-releasing nature of both the drugs in co delivery, may be
due to the interaction existing between the drugs (5-FU & CUR) which helps to protect them
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from degradation. Compared to the single delivery system, zero order kinetics occupies a major
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portion of the region for co-delivery system. It suggests that, it is an ideal delivery which refers
to the process of constant drug release from a drug delivery system (CS/Pd).
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The in vitro anti-proliferation effect of the nanoformulates against HT-29 cell lines were
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Briefly, cells were seeded in 96-well plates at the density of 1× 105 cells/well and incubated in a
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5% CO2 incubator for 72 hours. Then the cell culture medium was replaced by fresh medium
along with different concentration of drug loaded composites in 0.1% DMSO for 24h. Later,
Phosphate-buffered saline (pH ~7.4) was used to remove the samples. 20 µL per well
(5 mg mL-1) of MTT dye was added and incubated for 4h followed by the addition of 100 µL of
DMSO solvent. The absorbance of each sample was measured at a wavelength of 595 nm.
Untreated cells were considered as control. Inhibition of proliferation was calculated and the
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concentration required for 50% of viability (IC50) was found graphically. The relative cell growth
was compared with control cell and expressed as the cell viability (%), using the following
formula,
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Each experiment was carried out at least three times and statistical error analysis was estimated
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and is depicted in Fig. 9.
6.2. Cytotoxicity
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Fig. 9 shows the cytotoxicity analysis of drug loaded nanoformulations towards HT-29 cells.
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Analysis was performed for 24 h with various concentrations of drug encapsulated
nanoformulations to evaluate its inhibitory effect on cells. The conjugated nanoparticles show
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concentration dependent toxicity towards HT-29 cells. The half maximal inhibitory
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concentration (IC50) was found to be 18.3 µg/mL, 21.5 µg/mL and 14.6 µg/mL for 5-FU, CUR
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showed dose-dependent toxicity towards HT-29 cells but among them 5-FU+CUR encapsulated
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CS/Pd showed more efficiency than 5-FU encapsulated and CUR encapsulated CS/Pd. This is
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evident that dual drug loaded composite exhibits better performance which may be due to the
sustained release of 5-FU and CUR from the co-delivery platform [58].
7. Conclusion
Palladium embedded chitosan matrix nanocomposite was successfully synthesized by simple and
cost effective chemical reduction method. The prepared composites were analyzed using various
techniques. FTIR and XPS analysis confirmed that incorporation of palladium nanoparticles into
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the polymer which is due to the NH2 and OH groups of chitosan. HRTEM analysis revealed that
the palladium nanoparticles embedded in the matrix phase were spherical in shape with
approximate diameter of 3-4 nm. The obtained interplanar d-spacing value matches well with the
XRD pattern. Moreover, CUR and 5-FU were loaded in chitosan/Pd nanocomposite, individually
as well as in combination by simple chemical route. The XRD pattern confirms the formation of
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5-FU, CUR and 5-FU+CUR encapsulated CS/Pd nanocomposites. The increase in particle size
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of palladium and their agglomeration in the polymer matrix confirm the encapsulation of drug
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molecules into the composites. The possible drug molecule interaction was analyzed and
confirmed using FTIR and UV –Vis analysis. The entrapment and loading efficiency of CUR, 5-
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FU and 5-FU+CUR encapsulated CS/Pd nanocomposite was calculated. CUR and 5-FU release
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was analyzed using five mathematical models. Compared to the 5-FU, CUR monodelivery
system, on the basis of regression coefficient analysis, author concluded that mostly zero order
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kinetics occupies a major portion of the region for co-delivery system which clearly indicates the
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capability of the CS/Pd as a system for the sustained and prolonged release of 5-FU and CUR.
The cytotoxicity analysis of drug encapsulated nanocomposite towards human colorectal HT-29
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effect on growth of HT-29 cells over 5-FU, CUR monotherapy. In conclusion, dual drug loaded
nanoparticles of CS/Pd are promising platforms for the simultaneous release of multiple
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Acknowledgement
One of the authors S. D would like to thank UGC-UPE-Phase II for its financial assistance in the
form of fellowship to carry out this work successfully. The National Center for Nanoscience and
Nanotechnology, University of Madras is acknowledged for the XPS, HRTEM and FESEM
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facilities. SAIF, IIT Madras is acknowledged for FTIR measurements. Also author would like to
thank Dr. J. Senthilselvan, Asst. Professor, Department of Nuclear Physics, University of Madras
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Figure captions
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Figure 2: (a) XRD pattern of CS/Pd nanocomposites containing various percentages of palladium,
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and (b) drug (CUR, 5-FU and 5-FU + CUR) encapsulated nanocomposites.
Figure 3: (a) FTIR spectra of CS/Pd nanocomposites containing various percentages of palladium,
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(b) CUR and CUR encapsulated nanocomposite, (c) 5-FU and 5-FU encapsulated nanocomposite,
and (d) 5-FU and CUR encapsulated nanocomposite.
Figure 4: UV-Vis spectra of Palladium (II) acetate, CS/Pd and Inset: 5-FU (red), CUR (black) and
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dual (blue) loaded CS/Pd
Figure 5: (a, b) FESEM images of dual drug loaded nanocomposite and (c-e) its elemental mapping
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Figure 6: HRTEM images of CS/Pd represents the (a) polymer matrix nanocomposite, (b)
homogeneous distribution of palladium in CS, (c) diffraction planes of palladium, and (d) 5-FU and
CUR encapsulated nanocomposite showing the agglomeration of nanoparticles.
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Figure 7: XPS spectra of CS/Pd nanocomposite (a) survey spectrum, (b) C 1s spectrum, (c) N-1s
spectrum, (d) O 1s spectrum, and (e) Pd-3d spectrum
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Figure 8: Release profile of (a) CUR delivery from the nanocomposite, (b) 5-FU delivery from the
nanocomposite and (c) 5-FU and CUR co-delivery from composite
Figure 9: Cytotoxicity analysis of 5-FU encapsulated CS/Pd (CPF), CUR encapsulated CS/Pd
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Table 1. Encapsulation efficiency & Drug loading efficiency of drug loaded nanocomposites
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5-FU @ Chitosan/Pd 95.93 - 16.01 -
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CUR @ Chitosan/Pd - 96.33 - 18.19
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5-FU and CUR @ Chitosan/Pd 94.09 92.65 11.66 16.39
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Table 2. Mathematical models’ correlation coefficients for drug encapsulated nanoformulates
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Kinetics Region CS-Pd/5-FU CS-Pd/CUR CS-Pd/5-FU+CUR
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Graphical abstract
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Research highlights
(1) Chitosan/palladium nanocomposite was prepared for colon target dual drug delivery.
(2) Curcumin and 5-Flourouracil were loaded in chitosan/Pd nanocomposite, individually as well
as in combination by simple chemical route.
(3) Kinetics of single drug delivery was compared with co-delivery
(4) Co-encapsulated nanocomposite shows superior inhibitory effect on growth of HT-29 cells
over 5-FU, CUR monotherapy.
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