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

References 1. C.H. Koehne, and R.N. Dubois. Semin Oncol.31, 12-21(2004).

2. M. Rahman, K.Selvarajan, M. R. Hasan, A. P. Chan, C. Jin, J. Kim, S. K. Chan, N. D.

Le, Y.B. Kim, and I.T. Tai. Neoplasia 14, 624-633(2012).

3. K. Srimuangwong, C. Tocharus, P.Y.Chintana, A. Suksamrarn, and J. Tocharus. World J

Gastroenterol. 18, 2383-2389(2012).

4. H. Zhang, and X.F. Sun. Am J Gastroenterol 97, 1037-1041(2002).

5. N. Dhillon, B. B. Aggarwal, R. A. Newman, R. A. Wolff, A. B. Kunnumakkara, J. L.

Abbruzzese, C.S. Ng, V. Badmaev, and R. Kurzrock. Clin Cancer Res. 14, 4491-

4499(2008). 

6. K. Nakamura K, Y. Yasunaga, T. Segawa, D. Ko, J.W. Moul, S. Srivastava, and J S

Rhim. Int J Oncol. 21, 825-830(2002).  

7. S.H. Wu, L.W. Hang, J. S. Yang, H. Y. Chen, H.Y. Lin, J.H. Chiang, C. C. Lu, J. L.

Yang, T.Y. Lai T, Y.C. Ko, and J. G. Chung. Anticancer Res. 30, 2125-2134(2010).

8. J.J. Jeremy, and M. Hasan. Cancer Lett.255, 170-181(2007).

9. C.C. Su, J.G.Lin, T. M. Li, J.G.Chung, J. S. Yang, S.W. Ip, W. C. Lin, and G. W. Chen.

Anticancer Res. 26, 4379-4389(2006).

10. R. Hanif, L. Qiao, S.J. Shiff, and B. Rigas. J Lab Clin Med. 130, 576-584(1997).

11. A. Anitha, N. Deepa, K. P. Chennazhi, S. V. Nair, H. Tamura, and R. Jayakumar.

Carbohydr Polym. 83, 66–73(2011).

12. K. Srimuangwong, C. Tocharus, J. Tocharus, A. Suksamrarn, and P. Y. Chintana. World

J Gastroenterol. 18, 6951-6959(2012).

13. B. Du, L. Jiang, Q. Xia, and L. Zhong. Chemotherapy 52, 23-28(2006).

14. S. Carnesecchi, R. Bras-Goncalves, A. Bradaia, M. Zeisel, F. Gosse, and M. F. Poupon,

Raul. Cancer Lett 215, 53-59(2004).

15. L. Su, W. Anxun, J. Wenqi, and G. Zhongzhen. BMC Cancer 15,103-111(2008).

16. O. Raul, P. Jose, M. Consolacion, L.A. Jose, M.A.Ruiz, J. A. Pablo, C. Octavio, L.

Raquel, S. Ana, and A. Antonia. Int J Nanomed. 7, 95-107(2012).

17. A. Yan, J. Gu, Y. Guo, and D. Chen. The Pharm Soc Japan. 130, 801-804(2010).

18. Y. Zhang, J. Li, M. Lang, X. Tang, L. Li, and X. Shen. J Colloid Interface Sci. 354, 202-

209 (2011).

19. A. Gramont, A. Figer, M. Seymour, M. Homerin, A. Hmissi, J. Cassidy, C. Boni, H.

Cortes-Funes, A. Cervantes, G. Freyer, D. Papamichael, N.L. Bail, C. Louvet, D.

11

Hendler, F. de Braud C. Wilson, F. Morvan, and A. Bonetti . J Clin Oncol. 18, 2938-

2947(2000).

20. R.B. Diasio, and M.R. Johnson. Pharmacol 61, 199-203(2000).

21. J. Viguier, V. Boige, C. Miquel, M. Pocard, B. Giraudeau, J. C. Sabourin, M. Ducreux,

A. Sarasin, F. Praz. Clin Cancer Res. 11, 6212-6217(2005).

22. E. B. Yassin, M.K.Anwer, H. A. Mowafy, I.M.Elbagory, M.A. Bayomi, and I. A.

Alsarra. Int. J. Med. Sci. 7, 398-408(2010).

23. D. Yan, C. Chen, J. Gu, and J. Qin. J. Pharm. Pharmacol. 58, 1177-1186(2006).

24. S. Bisht, M. Mizuma,G. Feldmann, N.A. Ottenhof, S. M. Hong, D. Pramanik,V.Chenna,

C. Karikari, R.Sharma, M.G. Goggins, M.A. Rudek, R. Ravi, and A. Maitra. Mol Cancer

Ther. 9, 2255-2264(2010).

25. S. Acharya, and S. K. Sahoo. Adv Drug Deliv Rev. 63,170–183(2011).

26. Y. M. Tsai, C. F. Chien, L. C. Lin, and T. H. Tsai. Int J Pharm. 416,331-338(2011).

27. M.M.Yallapu, M.Jaggi, and S.C.Chauhan. Drug Discov Today. 17,71–80(2012).

28. B.S. Andreas, M. Hornof, T. Zoidl. Int J Pharm. 260, 229-37(2003).

29. S. Li, A.Wang, W.Jiang, Z. Guan. BMC Cancer (2008) 8:103 doi: 10.1186/1471-2407-8-

103.

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.