tamoxifen is anticancer drug which is used in treatment of breast cancer. but tamoxifen shows poor permeability and consequently efficacy. Recent studies showed an enhanced oral bioavailability of tamoxifen (TMX) by hydrophobically modified α-tocopherol succinate-g-carboxymethyl chitosan (Cmc-TS) micelles. As a continued effort, here we evaluated TMX-loaded polymeric micelles (TMX-PMs) for its enhanced permeability with increased anticancer efficacy and decreased hepatotoxicity. We employed co-solvent evaporation technique to encapsulate TMX into Cmc-TS. Apparent permeability assay of TMX-PMs was performed on Caco-2 cell line. The absorptive transport of TMX increased significantly about 3.8-fold when incorporated into Cmc-TS PMs. Cytotoxicity of Cmc-TS PMs was studied on MCF-7 cell line by MTT and; confocal microscopy was used for cellular uptake. Confocal microscopy revealed that Cmc-TS PMs could effectively accumulate in the cytosol of MCF-7 cell lines. In vitro data was further validated using N-methyl-N-nitrosourea (MNU)-induced mammary carcinogenesis model in Sprague-Dawley rats. Hepatotoxicity profiles of TMX-PMs at three different doses were also evaluated against the free drug TMX. TMX-PMs were more effective in suppressing breast tumor in MNU-induced mammary carcinoma model than free TMX with better safety profile. In addition, histological data shows that tumors are “benign” in TMX-PMs treated group compared with “malignant” tumors in free TMX treated and control groups. Overall, the results implicate that our Cmc-TS PMs may serve as a promising carrier for the intracellular delivery of anticancer drug molecules via oral route.

1. Presented by
:
Kiran (PE)
Aparna (BT)
Ajitha (PA)
Pathik (PC)

2. FLOW OF SEMINAR
INTRODUCTION
MATERIALS &
METHODS
PREPARATION
OF TMX-PMS
IN VITRO
PERMEABILITY
STUDY
CLSM
IN VITRO
CELLULAR
UPTAKE &
CYTOTOXIC
STUDY
IN VIVO
ANTITUMOR
EFFICACY &
HEPATOTOXICITY
STUDY
STATISTICAL
ANALYSIS
RESULT &
CONCLUSION

3. ABOUT THE ARTICLE
 AUTHORS : Sunil K. Jena, Sanjaya K. Samal, Shamandeep
Kaur, Mahesh Chand, Abhay T. Sangamwar.
 JOURNAL : European Journal of Pharmaceutical Sciences
 ACCEPTED DATE : 14 February 2017
 IMPACT FACTOR : 3.773
 PUBLISHER : ELSEVIER
 LAB: Department of Pharmaceutical Technology
(Formulations), b Department of Pharmaceutics, National
Institute of Pharmaceutical Education and Research,
Sector-67, S.A.S Nagar, Punjab-160062, India.

5. INTRODUCTION
Tamoxifen (TMX) is selective estrogen receptor modulator (SERM)
Tamoxifen shows high affinity towards estrogen receptor-positive tumors
It is considered as a first line therapy for early-stage breast cancer
treatment
On long term therapy its associated with an increased risk of endometrial
cancer and thromboembolic diseases
TMX undergoes extensive hepatic metabolism in humans, leading to the
formation of two active metabolites, 4-hydroxytamoxifen and endoxifen
(n-desmethyl-4-hydroxytamoxifen)

6. Moreover, these metabolites have been reported to exhibit a 100-fold
higher binding affinity to the estrogen receptor and are more
effective as cytoprotective agents than TMX
The overall anticancer efficacy of TMX has been reported to be
hampered by p-glycoprotein (p-gp) an efflux pump that is expressed
at the apical side of the intestinal membrane
Thus, there is a great interest in improving the oral
bioavailability of TMX by inhibiting the p-gp mediated TMX
efflux
CONTD….

7.  Several attempts have been made in the context of improving pharmacokinetic and
pharmacodynamic limitations of TMX
 The recent work includes polymeric micelles, polymeric nanoparticles, lipid based
nanoparticles, self-nanoemulsifying systems and TMX-phospholipid complex
 Amongst these, polymeric micelles, a self-assemble nanostructured drug
delivery system offers an advantage of;
a) higher drug loading
b) improved physical stability against the harsh conditions of gastro-intestinal (GI)
tract
c) being small in size, it can easily extravasate leaky tumor vasculature and reside
in the interstitial space near tumor cells for a longer period of time
CONTD….

8. MATERIALS
SUBSTANCES PURCHASED FROM
Tamoxifen (TMX). M/s Neon Laboratories Ltd.
(Mumbai, India).
Carboxymethyl chitosan (viscosity avg. M.W. ~
6.93 × 105 Da).
CMS Chemicals Ltd. (Berkshire, UK).
Coumarin-6, Verapamil, Hydrogen peroxide
(H2O2) solution (30% w/v in water).
Sigma-Aldrich Chemicals Pvt. Ltd.
(Bengaluru,India).
Ethylenediaminetetraacetic acid (EDTA),
Trichloroacetic acid (TCA), 5,5′-Dithiobis-(2-
nitrobenzoic acid) (DTNB), bisbenzimide
trihydrochloride glutathione, 5-Sulfosalicylic
acid, Hydroxylamine, 2-Thiobarbituric acid (TBA).
Hi-Media Laboratories (Mumbai,
India).
Dulbecco’s modified Eagle’s medium (DMEM),
trypsin-EDTA, penicillin (100 IU/ml),
streptomycin (100 µg/ml),
fetal bovine serum (FBS).
Biological Industries Israel Beit-
Haemek Ltd.
(Beit-Haemek, Israel).

9. CHARACTERISTICS OF TMX-PMS
Particle size - ~ 141.53 ± 2.59 nm
Polydispersity index- ~ 0.052 ± 0.02 .
Maximum TMX loading upto- 8.08 ± 0.98%.
Oral absorption - Via paracellular route.
Oral bioavailability- high
Inherent biodegradability and non-immunogenicity.

10. PREPARATION OF TMX-LOADED
POLYMERIC MICELLES

11. High M.wt
CMC
Low M.wt
CMC
HYDRO
LYSIS
Surface is
modified
with α-TS
Coupling
reagent
Amphiphilic
graft
co-polymer
CMC-TS
Hydroalcoholic
solution
Hydroalcoholic
phase
A
TMX Ethanol
Organic
phase
B

12. A B
SONICATION
20min ,
30±0.5ºC
Organic solvent removed
by rotavapor
Colloidal solution
Centrifuged
10,000 rpm
for 10 min
TMX-PM
Preparation of TMX-PM

13. IN VITRO PERMEABILITY STUDY

14. Caco-2 cells
DMEM , FBS , Penicillin and streptomycin
Conditions – 5% CO₂ , 37°c
Culture medium was renewed after every 2nd
day
Cell culture

15. Cells are
harvested by
trypsin-EDTA
Seeded apically
on
polycarbonate
membrane
Incubate at
37°C/5% CO₂
for 21 days
Growth
medium was
changed every
2-3 days
TEER was
measured
(Rм-Rв) ⅹ A
Once TEER value
was reached to
≥400Ω.cm² drug
permeability
assay was
performed
TEER Value measurement

16. TEER VALUE MEASUREMENT METHOD

17. Cells were
equilibrated in
HBSS for 30min
at 37°c
FOR A B
TMX,TMX-PM’s,
TMX-Verapamil in
HBSS with MES
2.5mM,pH6.5 were
added apically
Blank HBSS
medium containing
HEPES 5mM,pH
7.4 were added
basolaterally
Samples were
quantified using
reverse phase
HPLC with
photodiode array
detector
Cells were
incubated at 37°c
in 5% CO₂
For B A
TMX,TMX-
PM’s,TMX-
VERAPAMIL
added basolaterally
Drug permeability assay

18. CONTD….
APPARENT PERMEABILITY COEFFICIENT were
calculated by:
dQ/dt= cumulative transport rate,
C0=initial concentration of TMX in donor chamber,
A= surface area of membrane.
Papp=(dQ/dt)/(C0*A)

19. Results of permeability study

20. CONTD….
Fig. 1. Efflux ratio (Papp(B→A)/Papp(A→B)) of TMX, TMX with 100 µM
verapamil, and TMX-PMs.
Values are mean ± S.E.M (n = 4/time point). *Implies p < 0.0001 compared to TMX.

21. Cells seeded in 6
well plate
Grown by
incubating at
37°c/5% CO₂ for
15 days
Cell monolayer
incubated apically
with coumarin-6
loaded PM’s
In serum-free
DMEM with MES
2.5mM, pH 6.5 at
37°c/5% CO₂
Cells were rinsed
with phosphate
buffered saline pH
7.4
Cells observed
using CLSM
excited at 488nm
and emissions at
530nm.
Confocal laser scanning
microscopy(CLSM)

22. Results of CLSM
Fig. 2. CLSM image of Caco-2 cell monolayer after incubation with coumarin-6 loaded PMs
for 2 and 8 h. Left panel images represent Caco-2 cell monolayer with green fluorescence of
coumarin-6 loaded PMs, and right panel images represent images observed under phase
contrast microscopy. Scale bar represents 20 microns

23. IN VITRO CELLULAR UPTAKE
AND CYTOTOXICITY STUDY

24. DMEM supplemented with 10% FBS ,100 IU/ml penicillin
and 100µg/ml streptomycin at humidified atmosphere of 5%
CO2 at 37ºC
Human breast adenocarcinoma cell line
MCF-7
Cell culture

25. 1ml of culture medium is replaced with serum free
culture medium containing coumarin-6 PMs and
incubated for 1,4 & 24 Hrs
MCF-7 cells seeded at density 3x105
cells/well in 6 well plates
Preparation of coumarin-6 PMs
Cellular uptake studies

26. Cells were Observed in CLSM
Rinsed thrice with phosphate buffer
(pH 7.4,PBS)
Staining with Hoechst 33258 and
incubated for 10 mins
Contd…

27. Fig. 3. CLSM images of MCF-7 cells incubated with coumarin-6 loaded PMs for 1, 4 and 24
h. For each panel, images from left to right represent the cells with green fluorescence images
of coumarin-6 loaded PMs, the cells with nuclear staining by Hoechst 33258 (blue) and
images observed under phase contrast microscopy. Scale bar represents 20 microns.

28. Antiproliferative studies were performed with bare PMs at
similar concentrations
Culture media replaced with TMX & TMX-PMs at 6 different
concentrations(0.1, 0.5, 100, 500,1000µM)
Trypsinization of MCF-7 cell lines at density 5x103 cells/well in
96 well plates and incubated for 24 Hrs
Cytotoxic study

29. The absorbance of each well was taken at test wavelength
570nm and reference wavelength of 630nm
Optical density is measured using multi-mode microplate
reader
Incubated and subjected to MTT assay
Contd…
% Cell viability = (Intensity of the test sample/Intensity of the control) ×
100

30. Results
Fig. 4. In vitro cytotoxicity assay of (A) TMX and TMX-PMs, and (B) bared PMs
against MCF-7 cell lines.
Viability of cells as a function of TMX concentration was measured after an
incubation period of 24, 48 and 72 h by MTT assay. Results are expressed as mean ±
S.E.M (n = 4/time point).

31. At 50 Days All Rats Recieved Single Dose Of N-methyl
nitrosourease (MNU) [i.p.] (50mg/kg body weight)
MNU was dissolved immediately prior to its use in
0.9 % NaCl (pH = 4.0)
After 3 weeks of the injection rats were palpated
twice a week to check the presence of mammary
tumor
In vivo antitumor efficacy and
hepatotoxicity studies

32. Once the tumor volume reached approximately 100mm3
Rats were randomized into different groups
1st group
CONTROL
Only diet and
water for 60
days
2nd ,3rd ,4th group
Pure drug TMX
Once in a 3 days
(5mg ,7.5mg,10mg/Kg
for 60 days)
5th ,6th ,7th group
TMX-PMS
(5mg,7.5mg,10mg/kg
for 60 days)
Animals grouping

33. Tumor size digital vernier caliper (every 3days)
Tumor volume = ( a2 × b )/ 2
Here... a= smallest tumor diameter
b= largest tumor diameter
Relative tumor volume = ( Tx / To ) × 100
Where,
Tx = absolute tumor volume of the respective tumor on day X
To = absolute tumor volume of the respective tumor on day 0 when
treatment started
Estimation

34. Animals of all groups were sacrificed by
decapitation on the 61st day
Liver and tumor were excised
Liver
Liver homogenate was
prepared using ice-cold
phosphate buffer(pH=7.4,
10mM)
Used for determination of
oxidative stress
Tumor
 Avg. Tumor weight of each group
was measured and % tumor growth
inhibition was calculated
% tumor growth inhibition
=Avg. Tumor weight of treated group
Avg. Tumor weight of controlled group
Contd…

35. Liver and
mammary
tissues from
each group
Fixed in 10%
formaldehyde
for 1 week
Embedded in
paraffin
blocks
Cut into
7 μm slices
deparaffinizedhydrated
Stained with
haematoxylin
and eosin
Observed under Leica DMLP
reflected/ transmitted light
polarizing microscope
Histological examination

36. Fig. 5. (A) Representative picture of rats bearing a MNU-
induced mammary tumor (circled in
yellow lines) at day 61 post-treatment
Results of In vivo antitumor activity

37. (B) Change in relative tumor volume in rats receiving
TMX and TMX-PMs every third day for 60 days
Values are
mean ± S.E.M (n = 4). *Implies p < 0.05, †Implies p < 0.01.
Contd…

38. (C) Percent change in rat’s body
weight treated with TMX (5.0-10.0 mg/kg) and TMX-PMs (5.0-
10.0 mg/kg).
Contd…

39. Fig. 6. (A) Representative photographs of excised tumors (three from each
group) from rats
bearing MNU-induced mammary tumors at the end of 60 days treatment.
Contd…

40. B) The tumor growth inhibition rate was 38.2, 57.4 and 72.4% in the rats
treated with free TMX at doses 5.0, 7.5 and 10 mg/kg body weight
 While TMX-PMs >80% tumor growth inhibition
Contd…

41. H & E STAINING IMAGES OF
EXCISED TUMORS

42. FIG, 7A – 7C tumors from control group exhibited invasive ductal
adenocarcinomas of papillary and cribriform types
CF=cribriform
type
P=papillary
type
DR=desmoplastic
reaction
CC=cyst
component
SP=serrated
patterns
H & E STAINING IMAGES OF EXCISED TUMORS

43. Necrotic
material

44. Cribriform
type

45. Papillary
type

46. Considerable decrease in
cribriform patterns &
uniform size of nuclei

47. Considerably reduction of
cribriform/papillary patterns and
uniform nuclei size
However some cyst component(cc) &
Cell detritus(cd) were observed in core of
papillae

48. Tubular structures
separated only by
Small amount of
connective tissues

49. Individual tubule is
separated by
2 or more layers

50. Individual tubule is lined by
epithelial cells arranged in a
single layer
And has a
Secretion filled lumen

52. H & E STAINING OF LIVER
SECTIONS

53. Normal hepatocytes that are
Round or polygonal
With a
Small round nucleus and
Granular cytoplasm
H & E Staining of liver sections

54. Increased
sinusoidal spaces
H & E Staining of liver sections

55. Microvesicular
steatosis
H & E Staining of liver sections

56. Variable degree of
vacuolization
H & E Staining of liver sections

57. Normal hepatic architecture
Although there is a
slight sinusoidal dilation
And few vacuolated cytoplasm
H & E Staining of liver sections

58. STUDY OF LIVER ENZYMES

59. Table 2-Changes in the level of serum transaminases (ALT and AST),
TBARS and activity of liver oxidative enzymes (CAT and SOD) in
different groups under study.
Significant
restoration
compared to pure
TMX

60. 1.3 fold
1.6 fold
Table 2-Changes in the level of serum transaminases (ALT and AST),
TBARS and activity of liver oxidative enzymes (CAT and SOD) in
different groups under study.

61. 2 fold
1.8 fold
Table 2-Changes in the level of serum transaminases (ALT and AST),
TBARS and activity of liver oxidative enzymes (CAT and SOD) in
different groups under study.

62. Significant
restoration of
ALT and AST
level
Table 2-Changes in the level of serum transaminases (ALT and AST),
TBARS and activity of liver oxidative enzymes (CAT and SOD) in
different groups under study.

63. SOD SUPEROXIDE FREE RADICAL H2O2 REMOVED
CATDISMUTATION
Table 2-Changes in the level of serum transaminases (ALT and AST),
TBARS and activity of liver oxidative enzymes (CAT and SOD) in
different groups under study.

64. 2.1
fold
2.6
fold
Table 2-Changes in the level of serum transaminases (ALT and AST),
TBARS and activity of liver oxidative enzymes (CAT and SOD) in
different groups under study.

65. Table 2-Changes in the level of serum transaminases (ALT and AST),
TBARS and activity of liver oxidative enzymes (CAT and SOD) in
different groups under study.

66. Significant restoration
In levels of
Antioxidant enzymes
Table 2-Changes in the level of serum transaminases (ALT and AST),
TBARS and activity of liver oxidative enzymes (CAT and SOD) in
different groups under study.

67. CONCLUSION

69. CMC-PMs Increases the active transport of TMX across the caco-2 cell
monolayer, so that it easily enters to the blood stream and effectively reaches
the tumour tissues ,so that it leads to increase in bioavailability
Effective internalization in MCF-7 cells
TMX-PM more effective in suppressing breast tumour when compared with
free TMX moreover, it shows least toxicity Because there is a restoration of
hepatic enzymes
CMC-TS-PMs are biocompatible, low toxicity and promising oral drug
delivery system for poorly soluble anticancer drugs

70. ACKNOWLEDGEMENTS
We would like to acknowledge our respected faculties for helping us in
understanding the article and their support
WE WOULD ALSO LIKE TO THANK
Dr. Vinod Tiwari
Dr. Rakesh Tekade