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COMBINATORIAL APPROACH OF CURCUMIN AND
5-FLUOROURACIL LOADED CHITOSAN DERIVATIVES-BASED
NANOPARTICLES TOWARDS THE TREATMENT OF
CARCINOMA OF COLON
Synopsis of the thesis submitted to the
Amrita Vishwa Vidyapeetham University
for the award of the degree of
DOCTOR OF PHILOSOPHY
Submitted by
ANITHA A
(KH.NS.D*NMS09004)
Under the guidance of
DR. R. JAYAKUMAR
Amrita Center for Nanosciences & Molecular Medicine
Amrita School of Engineering
Amrita Vishwa Vidyapeetham University
Kochi (India)
DECEMBER 2013
AMRITA SCHOOL OF ENGINEERING
AMRITA VISHWA VIDHYAPEETHAM, KOCHI - 682041
AMRITA CENTRE FOR NANOSCIENCES AND MOLECULAR MEDICINE
DECLARATION
I Anitha A (KH.NS.D*NMS09004) hereby declare that this synopsis of the thesis titled
“Combinatorial Approach of Curcumin and 5-Fluorouracil Loaded Chitosan
Derivatives-Based Nanoparticles towards Treatment of Carcinoma of Colon’’ is a
bonafide work done under the guidance of Dr. R. Jayakumar, Professor, Amrita Centre
for Nanosciences and Molecular Medicine, Kochi and to the best of my knowledge and
belief it contains no materials previously published or written by another person, no
material which has been accepted for the award of any degree or diploma of the university
or other institute of higher learning, except where due acknowledgment has been made in
the text.
Place: Kochi Date: Signature of candidate
COUNTERSIGNED
Thesis Co-Guide Dr. Krishna Prasad Chennazhi Associate Professor Amrita Centre for Nanosciences & Molecular Medicine Amrita Institute of Medical Sciences and Research Centre, Kochi-682041
Thesis Guide Dr. R. Jayakumar Professor Amrita Centre for Nanosciences & Molecular Medicine Amrita Institute of Medical Sciences and Research Centre, Kochi-682041
1
Chapter 1: Introduction
Colorectal cancer is one of the major causes of cancer death worldwide and it
affects men and women equally. It is one among the 40% of all cases of cancer being
diagnosed yearly and third most leading cause of cancer death. The treatment
modalities for colon cancer include surgery, radiofrequency ablation, cryosurgery,
chemotherapy, radiation therapy and targeted therapy. Among which chemotherapy
utilizes different drugs or drug combination to reduce the cancer cell growth. The FDA
approved chemo drugs or drug combinations used in colon cancer include adrucil (5-
fluorouracil 5-FU), avastin (bevacizumab), camptosar (irinotecan hydrochloride),
capecitabine, cetuximab, eloxatin (oxaliplatin), erbitux (cetuximab) or drug
combinations such as capox, folfiri, folfiri-bevacizumab, folfiri-cetuximab, folfox,
zelox etc. In colon cancer, chemotherapy is given adjuvantly after surgery and the most
common chemo drug used is 5-FU. Being a thymidylate synthase inhibitor, it inhibits
the cancer cell growth through the arrest of cells in the S phase. The major drawback
of 5-FU is its systemic toxicity results from its non-specificity, low plasma half life
leading to the use of high doses and its inefficiency in chemo treatment results from
cyclooxygenase 2 (COX-2) over expression in colon cancer. The effectiveness of 5-FU
as a chemo drug can be improved by entrapping the drug in a polymer based carrier
nanoparticle system, wherein the drug being protected, reduces the non-selective
exposure and improves the plasma half life. In addition nanoparticles (NPs) have the
ability of enhanced permeability and retention effect (EPR) which helps in the
accumulation of drug loaded NPs in the tumour tissues in comparison with normal
tissues. Besides the efficacy of 5-FU as a chemo drug can be improved by a
combinatorial approach, where in 5-FU in combination with nontoxic COX-2
inhibitors such as curcumin (CRC) can be employed. There has been a reported study
for the potential enhancement of 5-FU anticancer efficacy with CRC under in vitro and
in vivo conditions through the inhibition of COX-2 gene/proteins.
Our research strategy is to explore the combinatorial anticancer effects of 5-
FU with CRC for improving the efficacy of 5-FU. The improvement in chemo
efficacy results from the CRC induced suppression of COX-2 expression, leads to
improved effectiveness of 5-FU in conventional chemotherapy. In the current work,
2
nanoencapsulation technique has been employed to improve the bioavailability of
both drugs and to reduce the non-selective exposure of 5-FU. The nanoencapsulation
has been made using chitosan derivatives based NPs having different chemical
functionality, N, O-carboxymethyl chitosan (N, O-CMC) and thiolated chitosan
(TCS). Thus the system1 (5-FU loaded N, O-CMC NPs / CRC-loaded N, O-CMC
NPs) and system 2 (5-FU loaded TCS NPs/CRC loaded TCS NPs) were made
separately and characterised. The in vitro combinatorial anticancer effects were
proved through different assays in colon cancer cells (HT29). The in vivo
pharmacokinetic results of system 1 and 2 in Swiss Albino mice model revealed the
improved plasma half life of both drugs unlike their bare counter parts.
Objectives of the study The specific objectives of the study include: 1. To develop and characterise N, O-carboxymethyl chitosan (N, O-CMC) and
thiolated chitosan (TCS) from chitosan.
2. To develop and characterise N, O-CMC and TCS Nanoparticles.
3. To evaluate the in vitro cytocompatibility and hemocompatibility of N, O-CMC
and TCS Nanoparticles.
4. To develop and characterise 5-FU loaded N, O-CMC nanoparticles (5-FU-N, O-
CMC NPs) and CRC loaded N, O-CMC nanoparticles (CRC- N, O-CMC NPs)
5. To evaluate the in vitro drug release, combinatorial anticancer efficacy of drug
loaded NPs (N, O-CMC system) towards colon cancer cells, in vivo pharmacokinetics
(PK) and biodistribution.
6. To develop and characterise 5-FU loaded thiolated chitosan nanoparticles (5-FU-
TCS NPs) and curcumin loaded thiolated chitosan nanoparticles (CRC-TCS NPs)
7. To evaluate the in vitro drug release, combinatorial anticancer efficacy of drug
loaded NPs (TCS system) towards colon cancer cells and in vivo pharmacokinetics
(PK) and biodistribution studies in Swiss Albino Mice model.
3
Literature Review
COX-2 protein over expression in colon cancer leads to ineffective
chemotherapy with 5-FU1. This problem can be solved by combining COX-2
inhibitors with chemodrugs.1 There has been reports showing the improvement in 5-
FU efficacy with aspirin,2 celecoxib,2 geraniol3 and genistein.3 Among these,
celecoxib is more effective in synergising the 5-FU efficacy in colon cancer but its
side effects limits its potential. CRC is known to have COX-2 inhibiting effects4 and
its anticancer potential towards many kinds of cancers were proven.5-10 The idea of
combinatorial anticancer effects of 5-FU with CRC against colon cancer was reported
earlier through the inhibition of COX-2 gene/protein under in vitro 11, 12 and in vivo
conditions.13 The combinatorial approaches with CRC makes more important because
of its wide anticancer potential and its safety in preclinical /clinical trials. Reports
were suggesting the potential of CRC as a suitable replacement for genistein and
geraniol and it can promotes 5-FU efficacy in various cancers including colon
cancer.14 5-FU triggers DNA mediated cell death in colon cancer by inhibiting the S
phase in cell cycle.15-23 Nanoencapsulation techniques being employed to enhance the
plasma half life of both CRC24-26 and 5-FU.15-18 In addition, chitosan and its
derivatives based nanoparticles were well exploited for drug delivery applications of
different drugs.17,27,28
Chapter 2: Materials and Methods
Synthesis and Characterisation of N, O-CMC, N, O-CMC Nanoparticles and In
vitro Evaluation
N, O-CMC was developed from chitosan through carboxymethylation reaction
using chloroacetic acid as the carboxymethylating agent. The resulting product was
characterised using FT-IR and degree of substitution was determined. N, O-CMC NPs
were obtained through ionic cross-linking reaction using pentasodium tripolyphosphate
(TPP). The resulting NPs were characterised using FT-IR, DLS, SEM and AFM. The
in vitro cytocompatibility (in colon cancer, HT 29 cells and normal cells, IEC6) and
hemocompatibility was evaluated.
4
Synthesis, Characterisation and In vitro Evaluation of CRC-N, O-CMC NPs and
5-FU-N, O-CMC NPs
5-FU-N, O-CMC NPs and CRC-N, O-CMC NPs were developed through the
ionic cross linking of TPP to the drug incorporated polymer solutions. Bovine serum
albumin (BSA) was used as the stabiliser to increase the aqueous dispersibility. The
resulting nanoparticle pellets were tested for its chemical composition (FT- IR),
entrapment and loading efficiency, in vitro drug release, hemocompatibility, cellular
uptake etc.
Evaluation of In vitro Combinatorial Anticancer Effects of CRC-N, O-CMC NPs
and 5-FU-N, O-CMC NPs
The combinatorial anticancer effects of the drug loaded NPs were quantified
through MTT, live dead, mitochondrial membrane potential and cell cycle analysis
measurements using the individual NPs and bare drugs in combination as controls.
Evaluation of In vivo Pharmacokinetics, Biodistribution and Histopathological
Assessment of CRC-N, O-CMC NPs and 5-FU-N, O-CMC NPs
The in vivo plasma concentration time profile of 5-FU-N, O-CMC NPs, CRC-
N, O-CMC NPs and co-administered 5-FU-N, O-CMC NPs and CRC-N, O-CMC NPs
were evaluated in Swiss Albino Mice using the bare drugs in combination and saline as
controls (72 hours). The pharmacokinetic profile was evaluated by HPLC using the
calibrated protocols up to 3 days. The organs of euthanized animals after 72 hours
were analyzed for biodistribution (through HPLC) and histopathologically assessed by
H and E staining.
Synthesis and Characterisation of TCS, TCS NPs and In vitro Evaluation
TCS was synthesised from chitosan using thioglycolic acid through EDC
mediated reactions. The reaction mixture was dialysed for a week and lyophilised. The
lyophilised product was characterised (FT-IR) and degree of thiolation was
determined. TCS NPs were developed through TPP mediated cross linking. The
5
resulting NPs were characterised using FT-IR, DLS, SEM, and zeta potential. The in
vitro cytocompatibility (HT29 and IEC6) and hemocompatibility was evaluated.
Synthesis, Characterisation and In vitro Evaluation of CRC-TCS NPs and 5-FU-
TCS NPs
CRC/5-FU (individually) incorporated TCS solutions (CRC in ethanol, 1mg/ml
for a ratio of 45ml 22.5 mg of TCS, 5mg of drug) were incubated with 0.1ml of 1%
BSA solution followed by cross-linking with TPP (1%) for 30minutes. Drug loaded
NPs was separated from the nanoparticle suspension by centrifuging the samples at
15000 rpm for 30 minutes. The resulting supernatant and pellet was collected and used
for further characterization and studies [in vitro drug release, hemocompatibility,
cellular uptake (rhodamine 123 labelled samples) etc].
Evaluation of In vitro Combinatorial Anticancer Effects of CRC-TCS NPs and 5-
FU-TCS NPs
The combinatorial anticancer effects of the drug loaded NPs were quantified
through MTT, live dead, mitochondrial membrane potential and cell cycle analysis
measurements using the individual nanoparticles and bare drugs in combination as
controls.
Evaluation of In vivo Pharmacokinetics, Biodistribution and Histopathological
Assessment of CRC-TCS NPs and 5-FU-TCS NPs
The in vivo plasma concentration time profile of 5-FU-TCS NPs, CRC-TCS
NPs and co-administered 5-FU-TCS NPs and CRC-TCS NPs were evaluated in Swiss
Albino Mice using the bare drugs in combination and saline as controls. The
pharmacokinetic profile was estimated through plasma using HPLC. The organs of
euthanized animals after 72 hours were analyzed for biodistribution (HPLC) and
histopathological assessment using H and E staining.
6
Chapter 3: Results and Discussion
N, O-CMC, TCS and its nanoparticles were synthesised, degree of
carboxymethylation (57 ± 8%) and thiolation (60 ± 5%) was determined and
characterised. The biocompatibility and hemocompatibility of the NPs were confirmed
(Fig. 1). 5-FU-N, O-CMC NPs, CRC-N, O-CMC NPs, 5-FU-TCS NPs and CRC-TCS
NPs were developed, and characterised (entrapment efficiency, loading efficiency and
in vitro hemocombatibility were proved). The in vitro drug release profile for all the
systems confirmed an initial burst release for the 5-FU system followed by a slow
release for 4 days. The CRC NPs system showed an initial slow release followed by a
sustained release profile up to 4 days. The in vitro combinatorial anticancer effects of
5-FU-N, O-CMC NPs, CRC-N, O-CMC NPs (system 1) and 5-FU-TCS NPs, CRC-
TCS NPs (system 2) were proved through MTT, live dead, cell cycle analysis and
mitochondrial membrane potential assays in HT 29 cells (for 48 hours, for a
concentration of 20 µM of CRC/ 20 µM of 5-FU within the NPs). For a concentration
of 20 µM of CRC and 20µM of 5-FU in the NPs, enhanced anticancer effects were
proven in HT29 unlike their individual counter parts and bare drugs. In conclusion, 5-
FU/CRC NPs in combination enhances the anticancer effects of 5-FU through the
inhibition of COX-2 protein which has been reported previously.
Fig. 1. (A) Schematic representation of N, O-CMC synthesis from chitosan, (B,C) SEM and
AFM images of N, O-CMC NPs, (D) cytocompatibility of N, O-CMC NPs through MTT assay
and (E, F) blood compatibility of N, O-CMC NPs through hemolysis and coagulation assays.
7
Table 1 Represents the characterisation data of 5-FU-N, O-CMC NPs, CRC-N, O-CMC NPs,
5-FU-TCS NPs and CRC-TCS NPs.
Fig. 2A Represents the cell death analysis through MTT measurements, where a, b, c, d, e, and
f represents 5-FU, CRC, combination of CRC and 5-FU, CRC-N, O-CMC NPs, 5-FU-N, O-
CMC NPs and the combination of both treated HT29 cells(48 hours). B, C, D, E, F, G and H
represents the live dead assay results, where B, C, D, E and F represents the control HT 29
cells, cells treated with CRC, 5-FU, combination of CRC and 5-FU, CRC-N, O-CMC NPs, 5-
FU-N, O-CMC NPs and the combination of both treated HT 29 cells, I and J represents the
quantification of combinatorial anticancer effects through mitochondrial membrane potential
(JC-1) and cell cycle analysis measurements (* indicates p< 0.05 compared to the samples).
8
Fig. 3. (A &C) the flow cytometric dot plots of quantification of combinatorial anticancer
effects of CRC-TCS NPs and 5-FU-TCS NPs through mitochondrial membrane potential(JC-
1) /cell cycle analysis measurements, where (a) control HT 29 cells, (b) treated with 5-FU,
(c)treated with CRC,(d) treated with a combination of 5-FU and CRC, (e)treated with 5-FU-
TCS NPs, (f) treated with CRC-TCS NPs, and (f) treated with a combination of CRC-TCS NPs
and 5-FU-TCS NPs after 48 hours. (B & D) represents the graphical representation of
mitochondrial membrane potential and cell cycle analysis measurements, where percentage
cell death was plotted against samples. (E & F) represents the pharmacokinetic profile of 5-
FU-TCS NPs and CRC-TCS NPs in Swiss Albino mice after 72 hours. (* indicates p< 0.05
compared to the samples).
The in vivo PK results of CRC-N, O-CMC NPs/5-FU-N, O-CMC NPs (system
1) and CRC-TCS NPs/5-FU-TCS NPs(system 2) individually and in combination
9
showed 28.6 ± 7.07 and 14.27 ± 4.00 µg/ml quantities respectively for 5-FU and CRC
in plasma up to 72 hours. The plasma concentration of 5-FU from a block copolymer
was reported earlier, and it showed a plasma concentration of 40-60 µg/ml of 5-FU up
to 48 hours.29 The system was proved to be inhibiting the growth of colon cancer
xenograft model.29 Even though our results of plasma concentration of 5-FU was
slightly lower, the combination of CRC/5-FU in the NPs can reduce colon cancer cell
growth through the COX-2 inhibiting activity of CRC, which can aid in improving the
anticancer efficacy of 5-FU. Hence the current systems of 5-FU/CRC in TCS NPs/N,
O-CMC NPs can produce combinatorial anticancer effects in colon cancer in which the
efficacy of 5-FU as a chemo drug could be enhanced and the dose reduction can be
achieved. For achieving this, the anticancer effects of the drug loaded NPs need to be
checked in colon cancer model models.
Chapter 4: Conclusions and Future Perspectives
Nanoformulations of 5-FU/CRC was developed using two chitosan derivatives based
nanoparticles with different chemical functionality ie., N, O-CMC and TCS to
overcome the low bioavailability of CRC/5-FU. The in vitro hemocompatibile nature
of 5-FU-N, O-CMC NPs/CRC-N, O-CMC NPs, and 5-FU-TCS NPs/CRC-TCS NPs
were proven. The in vitro combinatorial anticancer effects of 5-FU/CRC from the
CRC-N, O-CMC NPs/5-FU-N, O-CMC NPs and CRC-TCS NPs/5-FU-TCS NPs were
proved through different assays. The mechanism of enhanced anticancer effects could
be due to the inhibition of COX-2 by CRC, resulting in the improved anticancer effects
of 5-FU towards colon cancer as reported earlier. 3,12,13 Over all, the entrapped 5-
FU/CRC in N,O-CMC NPs and TCS NPs produced enhanced anticancer effects under
in vitro, and improved bioavailability under in vivo conditions. The in vivo plasma
concentration of 5-FU/CRC in the current experiment comes within the range where in
vitro combinatorial anticancer effects were proved. Furthermore, in clinical
application, the in vivo anticancer efficacy of the nanomedicine needed to be evaluated
in colon cancer.
10
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Carbohydr Polym. 83, 66–73(2011).
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Publications in International Journals
1. A. Anitha, V.V. Divya Rani, R. Krishna, V. Sreeja, N. Selvamurugan, S.V. Nair , H.
Tamura and R. Jayakumar, Synthesis, characterization, cytotoxicity and antibacterial
studies of chitosan, O-carboxymethyl and N, O-carboxymethyl chitosan nanoparticles.
Carbohydrate Polymers, 2009, 78, 672-677 (Impact factor: 3.5).
2. A. Anitha, N. Deepa, K. P. Chennazhi, S. V. Nair, H. Tamura and R. Jayakumar.
Development of mucoadhesive thiolated chitosan nanoparticles for biomedical
applications. Carbohydrate Polymers, 2011, 83, 66-73 (Impact factor: 3.5).
3. A. Anitha, K. P. Chennazhi, S. V. Nair and R. Jayakumar, 5-Flourouracil loaded N, O-
carboxymethyl chitosan nanoparticles as an anticancer nanomedicine for breast cancer.
Journal of Biomedical Nanotechnology, 2012, 8, 29-42. (Impact factor: 5.256).
4. A. Anitha, S. Maya, N. Deepa, K. P. Chennazhi, S. V. Nair, H. Tamura and R.
Jayakumar. Curcumin-loaded N, O-Carboxymethyl chitosan nanoparticles for cancer
drug delivery. Journal of Biomaterial Science Polymer Edition, 2012, 23, 1381-1400
(Impact factor: 1.7).
12
Awards
1. Best Poster Award for the poster titled “Nanocarrier based on Carboxymethyl chitosan
(N, O-CMC and O-CMC) for the delivery of curcumin to cancer cells” in Third
Bangalore Nano International Conference (2010).
Conferences Attended
1. ‘Nanocarrier based on carboxymethyl chitosan (N, O-CMC and O-CMC) for the delivery
of curcumin to cancer cells.’ A. Anitha, S. Maya, K. P. Chennazhi, S. V. Nair & R.
Jayakumar. Poster presentation in Third Bangalore Nano International Conference,
December 8 & 9, 2010, Held in Hotel Lalit Asok, Bangalore.
2. ‘Biodegradable chitosan derivative based nanoparticles as carrier for hydrophilic and
hydrophobic drug: development and characterization.’ A. Anitha, K. P.Chennazhi, S. V.
Nair & R. Jayakumar. Poster presentation in Amrita Bioquest International Conference
on Biotechnology for Innovative Applications, August 11-14, 2013, held in Amrita
VishwaVidyapeetham, Amritapuri Campus, Kerala.
3. ‘In Vitro Evaluation of 5-fluorouracil and curcumin loaded N, O-CMC nanoparticles for
colon cancer.’ A. Anitha, S. Maya, K. P. Chennazhi, S. V. Nair & R. Jayakumar.
Poster presentation in International Conference on Nanoscience and Nanotechnology,
January 20-23, 2012, held in Hyderabad India.