Accepted Manuscript

Title: Chitosan-Palmitic Acid Based Polymeric Micelles as

Promising Carrier for Circumventing Pharmacokinetic and
Drug Delivery Concerns of Tamoxifen

Authors: Nagarani Thotakura, Mukesh Dadarwal, Rajendra

Kumar, Bhupinder Singh, Gajanand Sharma, Pramod Kumar,
Om Prakash Katare, Kaisar Raza

PII: S0141-8130(17)30583-4
DOI: http://dx.doi.org/doi:10.1016/j.ijbiomac.2017.05.016
Reference: BIOMAC 7505

To appear in: International Journal of Biological Macromolecules

Received date: 15-2-2017

Revised date: 1-4-2017
Accepted date: 5-5-2017

Please cite this article as: Nagarani Thotakura, Mukesh Dadarwal,

Rajendra Kumar, Bhupinder Singh, Gajanand Sharma, Pramod Kumar, Om
Prakash Katare, Kaisar Raza, Chitosan-Palmitic Acid Based Polymeric
Micelles as Promising Carrier for Circumventing Pharmacokinetic and
Drug Delivery Concerns of Tamoxifen, International Journal of Biological
Macromoleculeshttp://dx.doi.org/10.1016/j.ijbiomac.2017.05.016

This is a PDF file of an unedited manuscript that has been accepted for publication.
As a service to our customers we are providing this early version of the manuscript.
The manuscript will undergo copyediting, typesetting, and review of the resulting proof
before it is published in its final form. Please note that during the production process
errors may be discovered which could affect the content, and all legal disclaimers that
apply to the journal pertain.
 Chitosan-Palmitic Acid Based Polymeric Micelles as Promising
Carrier for Circumventing Pharmacokinetic and Drug Delivery
Concerns of Tamoxifen

Nagarani Thotakura1#, Mukesh Dadarwal1#, Rajendra Kumar2,

Bhupinder Singh2, 3, Gajanand Sharma2, Pramod Kumar1, Om Prakash
Katare2 and Kaisar Raza1*

1Department of Pharmacy, School of Chemical Sciences and Pharmacy,

Central University of Rajasthan, Bandar Sindri, Dist. Ajmer, Rajasthan,
India-305 817.
2UGC-Centre of Excellence in Applications of Nanomaterials,
Nanoparticles and Nanocomposites, Panjab University, Chandigarh,
India-160 014.
3Division of Pharmaceutics, University Institute of Pharmaceutical
Sciences, Panjab University, Chandigarh, India-160 014.

# Both the authors contributed equally.

*Corresponding Author
Dr. Kaisar Raza
Department of Pharmacy
School of Chemical Sciences & Pharmacy
Central University of Rajasthan
Bandar Sindri, Distt. Ajmer, Rajasthan, India-305 817
E-mail: drkaisar@curaj.ac.in; razakaisar_pharma@yahoo.co.in
Abstract
Being a BCS class II drug and a good substrate for microsomal enzymes, tamoxifen
(TAM) offers a scope for research owing to poor aqueous solubility and
compromised bioavailability. The present study designs a novel copolymer derived
from palmitic acid and chitosan, and evaluate the derived TAM-loaded micelles for
various delivery attributes. The nanometric micellar carriers not only substantially
loaded the drug, but also controlled the rate of release of TAM. The designed
nanocarrier substantially enhanced the cytotoxicity of TAM on MCF-7 cancer cells.
The developed system was designed for intravenous route and was observed to be
substantially compatible enhancement of approx. 5 times in AUC vis-a-vis plain
drug and the biological half-life was increased by 3 times. The findings employing
new polymer-based carrier are promising in nature for the better delivery of similar
drugs.
Key words: Controlled release, MCF-7 cell line, Biocompatability, Bioadhesion, MTT
assay
1. Introduction

One of the leading causes for the deaths worldwide is cancer. Amongst all cancers,
breast cancer is considered as first cause for death of women worldwide [1]. There
are various drugs available for the treatment of breast cancer like docetaxel,
paclitaxel, doxorubicin and tamoxifen (TAM) [2]. TAM is one of the frequently
prescribed drugs [3]. As far as mechanism of action is concerned, TAM belongs to
class anti-estrogenic agents and for more than past two decades, it has been used for
the management of metastatic cancer and post-menopausal breast cancer [4],[5].
Even though it is used in the treatment of various cancers and psoriasis, the
therapeutic doses of the drugs are reported to cause side effects like pulmonary
embolism, endometrial cancer, ocular disturbances, distaste of food and abdominal
pain [6],[7],[8]. Though these concerns are inherited to drug, where drug delivery
approach may not be use in circumventing these concerns. Other challenges of TAM
like low aqueous solubility, high rate of drug metabolism and poor bioavailability
fall under the purview of drug delivery systems. In recent past, various groups have
attempted to improve the performance of TAM by means of novel materials and
carriers. A few instances are nanoparticles [9], lecithin organogels [5], liposomes [8]
and polymeric micelles [10].

A few attempts employing chitosan (CS) and derived polymer based nanocarriers
are also traceable. The biocompatability, immuno-neutrality and excellent
bioadhesion properties are the attributes of CS, which attract the researchers [11].
However, poor lipophilicity of this polymer is the main hurdle in its proper
utilization [12]. In order to improve the lipophilicity various moieties like PEG [13],
dextran [14], stearic acid [15] and PLGA [16] have been attached to this promising
polymer. However, no reports are available for the co-polymer conjugated with
palmitic acid (PA) and the subsequent exploration of resulting micelles in delivery of
TAM. The present study is an endeavour to chemically link PA with CS and study
the outcomes in the delivery of TAM to cancer cells.

2. Materials and methods

2.1. Materials

Tamoxifen (TAM) and dimethyl sulphoxide (DMSO) were purchased from M/s
Sigma Aldrich, New Delhi, India. Chitosan (degree of deacetylation ≥75%) was
obtained from M/s Himedia Laboratories Pvt Ltd., Mumbai, India. Sodium chloride
(NaCl), potassium dihydrogen phosphate (KH2PO4) and dipotassium hydrogen
phosphate (K2HPO4) and palmitic acid (PA) were supplied by M/s Central Drug
House (P) Ltd, New Delhi, India. Dialysis membrane was bought from M/s Himedia
Laboratories Pvt. Ltd., Mumbai, India. HPLC column (Oyster BDS C18 (25× 4.6 mm,
5µm) and membrane filters were purchased from M/s Merck Specialities Private
Limited, Mumbai, India. Ethanol was supplied by M/s Jai Chemical Pharma Works,
Jaipur. N, N’-dicyclohexyl carbodiimide (DCC), HPLC grade water, methanol and
acetonitrile (ACN) were obtained from M/s Spectrochem Pvt. Ltd., Mumbai, India.
MCF-7 cancer cell lines were procured from National Center for Cell Science, Pune,
India.

2.2. Synthesis of CS-PA Polymer

CS, 0.1 g was dissolved in 1mL of concentrated nitric acid, which was diluted with
20 mL of distilled water. PA (0.1g) and DCC (1g) were dissolved in 10 mL of ethanol
and this ethanolic solution of PA was added dropwise to CS solution with continous
stirring. The resultant solution was dialysed for 48 h against 1 L of distilled water.
The copolymer was harvested by evaporating the remaining solvent with the help of
rotary evaporator [17]. Figure 1 represents the synthetic scheme for preparation of
the desired copolymer.

2.3. Critical Micellar Concentration(CMC) Determination:

Well reported Iodine method was employed to determine the CMC values of the
copolymer. A standard solution was prepared by dissolving 250 mg of iodine and
500 mg of potassium iodide in 25 mL of distilled water. Various dilutions (1 to 16
µg/mL) of the copolymer were prepared and to each dilution, 25 µL of standard
iodine solution was added. The resulting solutions were kept overnight in dark and
analyzed at a wavelength of 366 nm using UV-Visible spectrophotometer (Cary 100
UV-Vis, M/s Agilent Technologies, Manesar, Haryana, India). From the results, a
graph was plotted between concentration of the copolymer and corresponding
absorbance. CMC value was the particular point where a sharp increase in
absorbance was observed [18].

2.4. Preparation of CS-PA-grafted TAM Polymeric Micelles:

TAM (10 mg) and copolymer (20 mg) were dissolved in 2 mL of DMSO. The solution
was filled in a syringe and added dropwise in a beaker containing 10 mL of
phosphate buffered solution, pH 7.4 (PBS) along with 0.2% w/w Tween 80. Water
bath sonication was performed for 1 h and the dispersion was subjected to dialysis
against distilled water for 24 h to remove the traces of organic solvent. Polymeric
micelles obtained were refrigerated for further use [12],[19].

2.5. Characterization Studies:

2.5.1. Fourier Transform Infrared Spectroscopy (FT-IR):

By using Fourier Transform Infrared Spectroscopy (FT-IR), structural aspects of the

copolymer were analyzed at initial stages. Potassium bromide punched compound
in the tablet form were scanned at the wave number range of 4000 to 400 cm-1
(Spectrum two, M/s Perkin Elmer Co., Waltham, Massachusetts, USA).

2.5.2. Nuclear Magnetic Resonance (NMR):

Amide bond formed between CS and PA was characterized using 1H NMR, which
was recorded using nuclear magnetic resonance spectrometer (Avance II 400 NMR
Spectrometer, M/s Bruker Bio Spin Corporation., Indiana, USA). Prepared polymer
was dissolved in deuterated chloroform (CDCl3) and scanned for NMR spectrum.

2.5.3. Particle Size Distribution and Zeta Potential:

Particle size, poly dispersity index (PDI) and zeta potential values of prepared
polymeric micelles were observed on using Malvern Zetasizer (M/s Malvern,
Worcestershire, UK) installed at Dr. S. S. Bhatnagar University Institute of chemical
Engineering & Technology, Panjab University, India. For these studies, polymeric
micelles were dispersed in PBS (1mg/mL) and used. Final result reported was
average of three consecutive readings (n=3).

2.5.4. Entrapment Efficacy (EE) and Drug Loading (DL) Studies:

Entrapment efficacy (EE) and drug loading (DL) were determined by diffusion
method [20]. The dialysis bag was filled with 1 mL of the nanodispersion and
dialysed against 25 mL of methanol for 2 h. The sink was analyzed for the
unentrapped drug. % EE and % DL were determined using equation (1) and (2), as
follows:

Theoretical drug − unentrapped drug (1)

%EE = × 100
Theoretical drug

(Theoretical drug − unentrapped drug) (2)

%DL = × 100
Amount of polymer used

2.5.5. In-vitro Drug Release Studies:

Both pure drug and drug-loaded polymeric micelles (equivalent to 1 mg of TAM)

were sealed into dialysis bags. These bags were suspended in 30 mL solution of
phosphate buffered solution, pH 5.6 and ethanol (9:1 v/v ratio) with continous
stirring [21]. Temperature was constantly maintained at 37±1°C. Sampling of 0.5 mL
was done at regular intervals maintaining sink conditions. Collected samples were
scanned using UV-Visible spectrophotometer at a wavelength of 301 nm (Cary 100
UV-Vis, M/s Agilent Technologies, Manesar, Haryana, India).

2.6. Evaluation Studies:

The animal protocols were duly approved by the Institutional Animal Ethics
Committee, Panjab University, Chandigarh, India.

2.6.1. Ex-vivo Hemolysis Studies:

Blood samples needed for this study was collected from unisex Wistar rats (200-250
g, 4-6 weeks old). Blood sample, harvested collected from retro orbital plexus of the
rat was collected in a test tube containing 124 mmol/L of sodium citrate solution.
Centrifugation was used for 5 min at 2000 × g, to separate the erythrocytes.
Erythrocytes were washed thrice with normal saline. The washed erythrocytes were
re-dispersed in 10 mL of normal saline. To this erythrocyte dispersion, both plain
drug and TAM-loaded polymeric micelles (equivalent to 1 mg of TAM) were added.
Reference sample used was erythrocytes dispersed in distilled water. All these test
tubes (samples and reference) were subjected to incubation for 1 h at 37°C. After this
1 h incubation all the sample and reference test tubes were centrifuged for 5 min at
2000 x g in order to attain transparent supernatant. This transparent was analyzed
using double beam spectrophotometer at wavelength (λmax) of 415 nm and %
hemolysis of the reference sample was considered as 100% [22].

2.6.2. In-vitro Cytotoxicity Assay:

To determine cytotoxicity potential of the prepared formulation, MTT assay was

preferred. MCF-7 human breast cancer cells were grown in 96 welled culture plates
using culture medium of EMEM (Eagles minimum essential medium) supplemented
with 10% FBS (fetal bovine serum) and 0.01 mg/mL of recombinant human insulin.
To these grown cells, known concentrations (1 µg/mL and 10 µg/mL) of various
samples (Pure TAM, blank polymeric micelles and TAM polymeric micelles) were
added and subjected to incubation at 37°C for 48 h. To the incubated system, 2.5
mg/mL of MTT solution was added and incubated for 4 h. After incubation plates
were centrifuged for 15 min at 400 ×g. DMSO, 150 µL was added to dissolve the
MTT-formazan crystals and optical density was measured at 570 nm and 620 nm
(reference wavelength) [23].

2.6.3. In-vivo Pharmacokinetic Studies:

Unisex Wistar rats of 4-6 weeks old were used for determination of various
pharmacokinetic parameters. There were two studied groups of 4 animals each,
comprising of Group 1 receiving plain TAM and Group 2 receiving TAM-loaded
polymeric micelles. Aseptically, 0.3 mL (5mg/kg of TAM) of test samples were
injected into rat through tail vein. Blood samples, 0.3mL were collected from retro-
orbital plexus at regular intervals and subjected to centrifugation (2000 × g) with
addition of 2 mL of methyl tert-butyl ether. Supernatant collected after
centrifugation was air dried and methanol was used for dissolving the obtained
residue. Prepared methanolic solution was analyzed using HPLC (LC-2010C HT,
M/s Schimadzu Co., Ltd., Chiyoda-ku, Tokyo, Japan) with the conditions as: Merck
HPLC Column: Oyster BDS C18 (250 x 4.6 mm, 5 µm); Detector used: PDA(SPD-
M20A); Mobile phase: acetonitrile and 50mM potassium phosphate, pH 3.0 (45:55%
v/v); Column temperature: 30°C; Flow rate: 2.0 mL/min; Injection volume: 5µL; Run
time: 30 min and Detection wavelength: 254 nm. Obtained data was fitted into one
compartmental open body i.v. push model [24]. Graph was plotted between log
concentration versus time (h), and various pharmacokinetic parameters were
calculated using Microsoft Office Excel [25].

3. Results and Discussion:

3.1. Characterization Studies:

Figure 2 represents FT-IR spectrum of CS-PA copolymer. From spectrum, there was
clear conformation of formation of amide bond as peaks at 3337 cm-1, 1660.4 cm-1 and
1078.7 cm-1 inferred the presence of (–N-H), (-C=O) and (-C-N) moieties,
respectively. On the other hand, peak at 1241.3 cm-1 inferred the (-C-O-) bonds
present in chitosan.

1H NMR spectrum of copolymer has been showcased in Figure 3. Like FT-IR, NMR
also provides strong evidence for formation of amide bond (-CO-NH-). Peaks
obtained at δ 3.13 represent the alkoxy group (R-CO-NH), δ 1.0 to 1.7 proved the
presence of protons of methyl groups (-CH3), and δ of 4.3, inferred the existence of
proton of amide (R-CO-N-H). Out of the used amount of palmitic acid, only 19.87%
of the fatty acid reacted with the chitosan, resulting in a copolymer comprising of
approx. two fatty acid molecules per chitosan molecule with an average mass of
around 5.5 KDa. The iodine method fetched with the CMC value of 4.6 µg/mL.
Similar copolymer have been reported to exhibit CMC in this range [15].

3.2. Particle Size Distribution and Zeta Potential:

Average particle size values of blank polymeric micelles and drug-loaded polymeric
micelles were observed as 67.43 ± 0.14 nm and 83.71 ± 0.15 nm respectively. The rise
in particle size may be ascribed to loading of TAM in the micellar structures [26].
PDI value for blank micelles was observed to be 0.21 and for drug loaded micelles
was found to be 0.25. PDI values less than 0.3 generally indicate the presence of
homogeneity in poly dispersed phase [27]. Due to presence of CS, the zeta potential
values of blank micelles and TAM-loaded micelles were observed as + 31.05 ± 0.11
mV and +37 ± 0.14 mV respectively, significantly positive in nature. Colloidal
dispersions with zeta potential values more than 25 mV are reported to be stable
[28]. These higher values of zeta potential assured the colloidal stability of the
developed systems.

3.3. Entrapment Efficiency (EE) and Drug Loading (DL):

Measured EE and DL values were observed as 93.76 ± 0.40 % and 10.24 ± 0.21 %
w/w respectively. Maximum entrapment was observed which may be ascribed to
hydrophobic core of CS-PA micelles. Even in 1:2 mass ratio of drug and polymer,
there was maximum amount of drug loading. Henceforth, this copolymer offers a
promise to load substantial amounts of chemotherapeutic agents, which is desired in
chemotherapy [29].

3.4. In-vitro Drug Release Studies:

Within 24 h of study period, drug release from bags of both TAM and polymeric
micelles was about 97.63 ± 0.96 % and 59.63 ± 1.35 %, respectively. Figure 4 shows
the drug release pattern from both pure drug and TAM-loaded micelles. Average
release flux of pure TAM and drug-loaded micelles were found to be 0.27 and 0.16
respectively. Better and sustained drug release can be ascribed to the amphiphilic
copolymer, CS-PA. However, the copolymer-based micelles released the loaded
drug in 48 h.

3.5. Ex-vivo Hemolysis Studies:

Figure 5 showcases the bar diagram representing % hemolysis of various test
formulations. From the figure, it was vivid that prepared copolymer was
biocompatible with RBCs, indicates the suitability of the same by i.v. route. As the %
hemolysis observed was less than 5 for all the systems, hence, they can be safe when
administered through intravenous route [24]. On the other hand, the hemotoxicity of
TAM was decreased by around 2-folds, after incorporation in the CS-PA micelles.
This might be plausibly due to the making of the direct drug contact with RBCs by
the surrounding nanocarrier molecules [30].

3.6. In-vitro Cytotoxicity Studies:

As shown in Figure 6, the microphotographs (from A to E) show that there was

gradual decrease in cell density, with increase in the concentration of TAM
employed. The superior performance of TAM-loaded micelles is clearly vivid. Figure
7 represents the bar graph, showing % growth inhibition of MCF-7 cells receiving
various treatments. Blank micelles showed negligible cytotoxicity, indicating non-
toxicity of copolymer. % growth inhibition from pure drug at 1 µg/mL and 10
µg/mL was found to be 15% and 33%, respectively. On the other hand, %
cytotoxicity was almost increased by 2 times by use of TAM-loaded micelles.
Enhanced permeation, endocytosis and better adhesion to neoplastic cells may be a
plausible reasons of substantial enhancement in cytotoxicity [17].

3.7. In-vivo Pharmacokinetic Studies:

Figure 8, shows the graph between time and plasma concentration of drug which is
collected at various time points. The observed plasma profile is characteristic of 1
CBM i.v. push. The decline in plasma concentrations in the group receiving plain
TAM was observed to be steeper than the group receiving TAM-loaded micelles.

Various pharmacokinetic parameters, calculated by incorporating the obtained

plasma data in one compartmental IV push model has been compiled in Table 1. It
was observed that AUC of the drug was increased by approx. 5 times by TAM-
loaded polymeric micelles, whereas elimination half life was increased by almost 2.5
times, and clearance was reduced by 1.7 times. The substantial increase in detectable
plasma concentrations can be ascribed to the incorporation of drug in interiors of
micelles, bypassing the microsomal enzymatic cascade [31]. Enhanced biological
residence and other pharmacokinetic attributes can be ascribed to the long
circulating nature of the micelles due to CS [17].

4. Conclusions:

The present work emphatically revealed the advantages of chitosan-palmitic acid

based polymeric micelles in developing controlled release system. The other
desirable outcomes were enhanced cytotoxic activity, hemo-compatibility and
improved pharmacokinetic profile. The strategic approach to enhance
biodistribution and bioadhesion by means of CS and enhanced drug loading by
virtue of PA, can offer an important drug delivery option in nanomedicine.

5. Acknowledgement:

The authors acknowledge the financial support from the University Grants
Commission, New Delhi, India in the form of UGC-Start-Up Grant (F.30-
18/2014BSR) to the corresponding author.

6. Conflicts of Interest: The authors report no conflict of interest.

7. References:

[1] E. Memisoglu-Bilensoy, I. Vural, A. Bochot, J.M. Renoir, D. Duchene, A.A.

Hincal, Tamoxifen citrate loaded amphiphilic beta-cyclodextrin nanoparticles:
in vitro characterization and cytotoxicity., J. Control. Release. 104 (2005) 489–
96.

[2] P. Kumar, K. Raza, L. Kaushik, R. Malik, S. Arora, O.P. Katare, Role of

Colloidal Drug Delivery Carriers in Taxane-mediated Chemotherapy: A
Review., Curr. Pharm. Des. (2016).

[3] A. Bhatia, B. Singh, S. Bhushan, O.P. Katare, Tamoxifen-encapsulated vesicular

systems: cytotoxicity evaluation in human epidermal keratinocyte cell line.,
 Drug Dev. Ind. Pharm. 36 (2010) 350–4.

[4] B. Sahana, K. Santra, S. Basu, B. Mukherjee, Development of biodegradable

polymer based tamoxifen citrate loaded nanoparticles and effect of some
manufacturing process parameters on them: a physicochemical and in-vitro
evaluation., Int. J. Nanomedicine. 5 (2010) 621–30.

[5] A. Bhatia, B. Singh, K. Raza, S. Wadhwa, O.P. Katare, Tamoxifen-loaded

lecithin organogel (LO) for topical application: Development, optimization and
characterization., Int. J. Pharm. 444 (2013) 47–59.

[6] V.C. Jordan, Tamoxifen: a most unlikely pioneering medicine., Nat. Rev. Drug
Discov. 2 (2003) 205–13.

[7] C.K. Osborne, Tamoxifen in the treatment of breast cancer., N. Engl. J. Med.
339 (1998) 1609–18.

[8] A. Bhatia, R. Kumar, O.P. Katare, Tamoxifen in topical liposomes:

development, characterization and in-vitro evaluation., J. Pharm. Pharm. Sci. 7
(2004) 252–9.

[9] C. Altmeyer, T.K. Karam, N.M. Khalil, R.M. Mainardes, Tamoxifen-loaded

poly(L-lactide) nanoparticles: Development, characterization and in vitro
evaluation of cytotoxicity, Mater. Sci. Eng. C. 60 (2016) 135–142.

[10] G. Cavallaro, L. Maniscalco, M. Licciardi, G. Giammona, Tamoxifen-loaded

polymeric micelles: preparation, physico-chemical characterization and in
vitro evaluation studies., Macromol. Biosci. 4 (2004) 1028–38.

[11] M. Prabaharan, J.F. Mano, Chitosan-Based Particles as Controlled Drug

Delivery Systems, Drug Deliv. 12 (2008) 41–57.

[12] F.-Q. Hu, G.-F. Ren, H. Yuan, Y.-Z. Du, S. Zeng, Shell cross-linked stearic acid
grafted chitosan oligosaccharide self-aggregated micelles for controlled release
of paclitaxel., Colloids Surf. B. Biointerfaces. 50 (2006) 97–103.

[13] N. Bhattarai, H.R. Ramay, J. Gunn, F.A. Matsen, M. Zhang, PEG-grafted

chitosan as an injectable thermosensitive hydrogel for sustained protein
 release., J. Control. Release. 103 (2005) 609–24.

[14] S. Paramasivan, D. Jones, L. Baker, L. Hanton, S. Robinson, P.J. Wormald, L.

Tan, The use of chitosan-dextran gel shows anti-inflammatory, antibiofilm,
and antiproliferative properties in fibroblast cell culture., Am. J. Rhinol.
Allergy. 28 (2014) 361–5.

[15] N. Thotakura, M. Dadarwal, P. Kumar, G. Sharma, S.K. Guru, S. Bhushan, K.

Raza, O.P. Katare, Chitosan-Stearic Acid Based Polymeric Micelles for the
Effective Delivery of Tamoxifen: Cytotoxic and Pharmacokinetic Evaluation.,
AAPS PharmSciTech. (2016). doi:10.1208/s12249-016-0563-6.

[16] C.K. Thakur, N. Thotakura, R. Kumar, P. Kumar, B. Singh, D. Chitkara, K.

Raza, Chitosan-modified PLGA polymeric nanocarriers with better delivery
potential for tamoxifen., Int. J. Biol. Macromol. 93 (2016) 381–389.
doi:10.1016/j.ijbiomac.2016.08.080.

[17] Y.-T. Xie, Y.-Z. Du, H. Yuan, F.-Q. Hu, Brain-targeting study of stearic acid-
grafted chitosan micelle drug-delivery system., Int. J. Nanomedicine. 7 (2012)
3235–44.

[18] S.K. Hait, S.P. Moulik, Determination of critical micelle concentration (CMC)
of nonionic surfactants by donor-acceptor interaction with lodine and
correlation of CMC with hydrophile-lipophile balance and other parameters of
the surfactants, J. Surfactants Deterg. 4 (2001) 303–309. doi:10.1007/s11743-001-
0184-2.

[19] T.G. Kim, H. Lee, Y. Jang, T.G. Park, Controlled release of paclitaxel from
heparinized metal stent fabricated by layer-by-layer assembly of polylysine
and hyaluronic acid-g-poly(lactic-co-glycolic acid) micelles encapsulating
paclitaxel., Biomacromolecules. 10 (2009) 1532–9. doi:10.1021/bm900116r.

[20] M. Jones, J. Leroux, Polymeric micelles - a new generation of colloidal drug

carriers., Eur. J. Pharm. Biopharm. Off. J. Arbeitsgemeinschaft Für Pharm.
Verfahrenstechnik e.V. 48 (1999) 101–11.

[21] H. Yadav, P. Kumar, V. Sharma, G. Sharma, K. Raza, O.P. Katare, Enhanced

 efficacy and a better pharmacokinetic profile of tamoxifen employing
polymeric micelles, RSC Adv. 6 (2016) 53351–53357. doi:10.1039/C6RA10874A.

[22] K. Raza, N. Kumar, C. Misra, L. Kaushik, S.K. Guru, P. Kumar, R. Malik, S.

Bhushan, O.P. Katare, Dextran-PLGA-loaded docetaxel micelles with
enhanced cytotoxicity and better pharmacokinetic profile., Int. J. Biol.
Macromol. 88 (2016) 206–212.

[23] K. Raza, D. Kumar, C. Kiran, M. Kumar, S.K. Guru, P. Kumar, S. Arora, G.

Sharma, S. Bhushan, O.P. Katare, Conjugation of docetaxel with multiwalled
carbon nanotubes and co-delivery with piperine: Implications on
pharmacokinetic profile and anti-cancer activity., Mol. Pharm. 13 (2016) 2423–
32.

[24] K. Raza, N. Thotakura, P. Kumar, M. Joshi, S. Bhushan, A. Bhatia, V. Kumar, R.

Malik, G. Sharma, S.K. Guru, O.P. Katare, C60-fullerenes for delivery of
docetaxel to breast cancer cells: A promising approach for enhanced efficacy
and better pharmacokinetic profile., Int. J. Pharm. 495 (2015) 551–9.

[25] L. Shargel, A.B.C. Yu, Applied Biopharmaceutics and Pharmacokinetics,

McGraw-Hill Professional Publishing, 1999.

[26] P. Ramalingam, T. Ko, Y, Improved oral delivery of resveratrol from N-

trimethyl chitosan-g-palmitic acid surface-modified solid lipid nanoparticles.,
Colloids Surf. B. Biointerfaces. 139 (2016) 52–61.

[27] M. Gaumet, A. Vargas, R. Gurny, F. Delie, Nanoparticles for drug delivery: the
need for precision in reporting particle size parameters., Eur. J. Pharm.
Biopharm. 69 (2008) 1–9.

[28] Y. Hu, X. Jiang, Y. Ding, H. Ge, Y. Yuan, C. Yang, Synthesis and

characterization of chitosan–poly(acrylic acid) nanoparticles, Biomaterials. 23
(2002) 3193–3201.

[29] J. Della Rocca, D. Liu, W. Lin, Are high drug loading nanoparticles the next
step forward for chemotherapy?, Nanomedicine (Lond). 7 (2012) 303–5.
doi:10.2217/nnm.11.191.
[30] V.R. Muzykantov, Drug delivery by red blood cells: vascular carriers designed
by mother nature., Expert Opin. Drug Deliv. 7 (2010) 403–27.
doi:10.1517/17425241003610633.

[31] A.K. Jain, S. Jain, A.C. Rana, Metabolic enzyme considerations in cancer
therapy., Malays. J. Med. Sci. 14 (2007) 10–7.

Figure 1: Synthetic scheme for preparation of CS-PA copolymer

Figure 2: FT-IR spectrum of CS-PA co-polymer
 Figure 3: NMR spectrum of CS-PA copolymer

Figure 4: Graph showing the release pattern of pure TAM and TAM-loaded
polymeric micelles
 Figure 5: Bar diagram showing % hemolysis

Figure 6: Microphotographs of MCF-7 cells receiving various treatments (a) Control;

(b) TAM (1 µg/mL); (c) TAM (10 µg/mL); (d) TAM-loaded micelles (1 µg/mL); and
(e) TAM-loaded micelles (10 µg/mL.
Figure 7: Bar graph showing % growth inhibition of MCF-7 cells after receiving
various treatments.

Figure 8: Time versus plasma concentration plot

Table 1: Various pharmacokinetic parameters obtained by employing the obtained
data in IV push

Parameter Pure TAM TAM-loaded micelles

[AUC] 0-∞ (µg mL-1h) 16.45 78.50
K (h-1) 0.19 0.08
Cl (mL/h) 10.22 5.77
t½ (h) 3.54 8.60
Vd (L) 1.96 0.46