Accepted Manuscript

In vitro cytotoxicity study of dual drug loaded chitosan/palladium

nanocomposite towards HT-29 cancer cells

S Dhanavel, E A K Nivethaa, V Narayanan, A Stephen

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
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authors. Please check if appropriate. Msc(2016), doi: 10.1016/j.msec.2017.03.058

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 ACCEPTED MANUSCRIPT

In vitro cytotoxicity study of dual drug loaded chitosan/palladium

nanocomposite towards HT-29 cancer cells
S Dhanavela, E A K Nivethaaa, V Narayananb and A Stephena,*
a
Material Science Centre, Department of Nuclear Physics, University of Madras, Guindy Campus, Chennai-25,
India.
b
Department of Inorganic Chemistry, University of Madras, Guindy Campus, Chennai 600 025, India.

*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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inhibiting the growth of cancer cells.

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Keywords: Curcumin, 5-Fluorouracil, Chitosan/Pd, co-delivery, HT-29 cells

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

synergistic anticancer efficacy [9, 10].

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Nanotechnology is a rapidly growing research domain in the field of catalyst, biosensors, bio-
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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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metal nanoparticles which makes them eco-friendly.

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Chitosan (CS) is an abundant linear cationic polysaccharide, which consists of randomly

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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physical properties [24]. Its abundant availability, biodegradability, biocompatibility, non-

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 such as anti-inflammatory, antimicrobial, antitumor, antiparasitic and antioxidant

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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

loaded (individually) N,O-carboxymethyl chitosan nanoparticles towards the inhibition of colon

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,

encapsulating a combination of drugs (CUR and 5-FU) in chitosan/palladium is expected to

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

colon cancer cells.

2. Materials and Methods

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.

2.2. Characterization details

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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

spectrophotometer Analytik Jena specord 200 plus.

2.3. Fabrication of chitosan/palladium nanocomposite

Chitosan/palladium nanocomposite was synthesized using a simple, cost effective chemical

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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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was obtained and characterized.

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2.4. Synthesis of drugs encapsulated chitosan/palladium nanocomposite

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The chitosan/palladium-5% nanocomposite is used as a nanocarrier for the dual drug

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.

2.5. Cell lines and culture conditions

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Human colon tumor cell line, HT-29 cells were obtained from National Centre for Cell Sciences
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(NCCS), Pune, India. The cells were cultured in Minimal essential media supplemented with
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10% FBS and 1% penicillin-streptomycin solution and incubated in an incubator containing 5%

CO2 at 37 °C.
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3. Results and Discussion:

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3.1. Powder XRD

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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

5-FU and CUR are given in supplementary information (Fig. S2).

3.2. FTIR analysis

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Fig. 3a shows the FT-IR spectra of the composites containing different weight percentage of
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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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been encapsulated well into the chitosan matrix.

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3.3. UV-Vis analysis

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

plasmon absorbance) was ascribed by Haizhen Huang et. al [42].

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

nanocomposite. The characteristic absorbance peak of 5-FU in 5-FU encapsulated CS/Pd is at

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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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CS/Pd nanocomposite [43].

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3.4. Electron microscopy (SEM and TEM) and elemental mapping

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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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3.5. X-ray photoelectron spectroscopy analysis

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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.

4. Drug entrapment efficiency and Drug loading efficiency of drugs

Drug loading and entrapment efficiency of CS/Pd nanocomposite was calculated as follows

(Total 5-FU or CUR) – Free (5-FU or CUR)

Drug entrapment efficiency = ------------------------------------------------------- X 100 (1)
(Total 5-Fu or CUR)
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(Total 5-FU or CUR) – Free (5-FU or CUR)

Drug loading efficiency = -------------------------------------------------------------- X 100 (2)
Weight of 5-FU or CUR loaded nanocomposite taken

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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as well as 5-FU and CUR loaded N,O- carboxymethyl chitosan nanoparticles by

A. Anitha et al [36].

5. In vitro drug release profile

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

chitosan, thereby facilitating the diffusion of drug [53].

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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

determination of drug release mechanism in colon cancer therapy. A calibration graph of

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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

calibration curve. Similarly dual drug release was evaluated.

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.

Zero order model

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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1001/3 – [100 - (Mt/M∞)]1/3 = Kc t (6)

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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

Case II transport mechanism, suggesting that relaxation of hydrophilic polymer chains is

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

considered mathematical models.

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5.1. Curcumin delivery kinetics
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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

transport of Korsmeyer-Peppas which suggest the Fickian diffusion (n = 0.206) process.

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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

kinetic model with the Fickian diffusion (n = 0.098).

5.2. 5-FU delivery kinetics

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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.

5.3. Comparison of 5-FU and Curcumin delivery

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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6. In vitro cytotoxicity study

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6.1. Cytotoxicity assay

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The in vitro anti-proliferation effect of the nanoformulates against HT-29 cell lines were
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evaluated by MTT assay (3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide).

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,

(A595 of treated cells)

Cell viability (%) = ------------------------------------ (8)
(A595 of control cells)

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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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and 5-FU+CUR encapsulated nanoformulations respectively. All the prepared formulations

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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cells was analyzed. In particular, co-encapsulated nanocomposite shows superior inhibitory

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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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therapeutic agents for the achievement of effective therapy.

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

for the UV-Vis Measurements.

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Figure captions

Figure 1: Schematic representation of the formation of 5-FU/CUR loaded Chitosan/Pd

nanocomposite.

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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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(CPC) and 5-FU + CUR encapsulated CS/Pd (CPFC)

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Table 1. Encapsulation efficiency & Drug loading efficiency of drug loaded nanocomposites

Sample Encapsulation Drug Loading efficiency

efficiency (%) (%)
5-FU CUR 5-FU CUR

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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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5-FU@ co-delivery CUR @ co-delivery

Zero I 01 to 12 h = 0.9035 01 to 09 h = 0.9122 01 to 06 h = 0.9258 01 to 18 h = 0.9827
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order II 15 to 44 h = 0.8962 12 to 32 h = 0.9736 09 to 44 h = 0.9216 22 to 39 h = 0.9809

III 50 to 72 h = 0.8946 36 to 72 h = 0.9564 50 to 72 h = 0.9645 44 to 72 h = 0.9045
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First I 01 to 12 h = 0.8419 01 to 09 h = 0.9066 01 to 06 h = 0.8810 01 to 18 h = 0.9718

order II 15 to 44 h = 0.8810 12 to 32 h = 0.9860 09 to 44 h = 0.9147 22 to 39 h = 0.9807
III 50 to 72 h = 0.8731 36 to 72 h = 0.9462 50 to 72 h = 0.9595 44 to 72 h = 0.8969
CE

Kaursm I 01 to 12 h = 0.9396 01 to 09 h = 0.9145 01 to 06 h = 0.8545 01 to 18 h = 0.9073

eyer- II 15 to 44 h = 0.9282 12 to 32 h = 0.9389 09 to 44 h = 0.9022 22 to 39 h = 0.9551
Peppas III 50 to 72 h = 0.9130 36 to 72 h = 0.9854 50 to 72 h = 0.9729 44 to 72 h = 0.9350
AC

Hixon - I 01 to 12 h = 0.9307 01 to 09 h = 0.9142 01 to 06 h = 0.9244 01 to 18 h = 0.9806

Crowel II 15 to 44 h = 0.9110 12 to 32 h = 0.9637 09 to 44 h = 0.9190 22 to 39 h = 0.9805
III 50 to 72 h = 0.9565 36 to 72 h = 0.9663 50 to 72 h = 0.9711 44 to 72 h = 0.9079

Higuchi I 01 to 12 h = 0.9567 01 to 09 h = 0.9490 01 to 06 h = 0.9072 01 to 18 h = 0.9746

II 15 to 44 h = 0.9191 12 to 32 h = 0.9470 09 to 44 h = 0.9178 22 to 39 h = 0.9701
III 50 to 72 h = 0.9135 36 to 72 h = 0.9765 50 to 72 h = 0.9696 44 to 72 h = 0.9230
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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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