This is the our original work which i have done in my M.Phil course work as a part of dissertation.

1. BOVINE SERUM ALBUMIN NANOPARTICLE FOR
THE EFFICIENT DELIVERY OF ANTI-CANCER DRUG
(BERBERINE):
PREPARATION, CHARACTERIZATION AND IN VITRO STUDIES
SUPERVISOR
Dr. Sunita Patel
Assistant Professor
School of Life Sciences
Central University of Gujarat
PRESENTED BY
Raghu Solanki
Enrolment No. 170312005
School of Life Sciences
Central University of Gujarat
M.Phil. Dissertation work
presentation

2. LAYOUT OF MY PRESENTATION
OVERVIEW
HYPOTHESIS
OBJECTIVES
METHODOLOGY
RESULTS
CONCLUSION
REFERENCES
ACKNOWLEDGMENT

3. 24.20%
8.40%
5.10%
4.10%
9.50%6.60%
4.40%
37.70%
Estimated number of cancer cases
in women
Breast Lung Thyroid
stomach colorectum cervix
corpus uteri others
15.80%
13.80%
9.50%
7.50%
6.50%5.60%
4.90%
37.70%
Estimated number of deaths in
women
Breast Lung Colorectum
Cervix uteri Stomach Liver
Pancreas Others
Data source: Globocan 2018
CANCER: Cancer is a generic term for a large group of diseases
characterized by the growth of abnormal cells. That can invade other
parts of the body and spread to other organs (WHO)
OVERVIEW

4. Breast cancer usually begins in the ducts that carry milk
to the nipple or within the smaller structures of the lobes.
Breast cancer can spread to other parts of the body via the
lymphatic system or the bloodstream.
Where does breast cancer originate?
Breast Cancer: Uncontrolled growth of breast cells.
Breastcancer
Based on Tissues
Ductal
carcinoma
Lobular
carcinoma
Based on Hormones
and Genes
Luminal A
Luminal B
HER2 Positive
Triple
Negative
Based on
Invasiveness
Invasive
Noninvasive

5. Treatment
Local
Surgery
Radiation
Systemic
Chemotherapy
Hormone
therapy
Targeted
therapy
Natural plant extract
Berberine [BBR]

6. BERBERINE
Anti-
Inflamm
atory Anti
hyperten
sive
Anti
depressant
Anti-
cancer
Hepato
protective
Hypo
lipidemic
Neuro
protective
Anti-
Microbial
Anti
Diarrheal
Anti
Diabetic
Anti-
oxidant
BERBERINE
Solutions
(Liu et al., 2016)
Drug delivery system

7. Albumin is the natural and most abundant
(~59%) plasma protein and has high affinity to
bind drug ligands and metabolites, thus, acting as
a carrier for them.
Albumin is a monomer, has 3 domains (Domain
I, II and III) which further composed of 2 sub-
domains A and B.
The drug binding site is present in sub-domain
IIA and IIIA.
 Biocompatible
 Biodegradable
 Non-immunogenic
 Non-toxic
Albumin Protein as a drug nanocarrier..!!
Physiological properties
PDB: 3V03

8. HYPOTHESIS
Berberine loaded BSA nanoparticles may be an
efficient drug delivery vehicle, which will emerge
as a novel therapeutic approach for the treatment of
breast cancer.

9. OBJECTIVES
1
• Preparation and purification of blank BSA
nanoparticles (BSA NPs) and Berberine loaded BSA
Nanoparticles (BBR-BSA NPs).
2
• Characterization of BSA NPs and BBR-BSA NPs.
3
• Optimization of encapsulation efficiency, Loading
capacity and in vitro release study of prepared
nanoparticles
4
• In vitro cytotoxic studies of prepared BBR-BSA NPs

10. METHODOLOGY
DLS
SEM
FTIR
DSC
TGA
MTT Assay
Trypan Blue Assay
Apoptosis Study
Cellular Uptake study
 EE %
 LC %
 % Cumulative Drug Release
 Nanoparticles stability

11. RESULTS & DISCUSSION

12. OBJECTIVE: 1
PREPARATION AND PURIFICATION OF BSA
NANOPARTICLES (BSA NPS) AND BERBERINE LOADED
BSA NANOPARTICLES(BBR-BSA NPS)

13. 200 mg
BSA
dissolved
in 2 mL
Milli Q
pH adjusted to 8.5
20 mg BBR dissolved
in 8 ml of Ethanol
BBR solution
addeddrop wise (1
mL/min) with constant
stirring (550 rpm) in
BSA solution
235 µl
8%
Glutaraldehyde
added
Stabilized for 24 h
(room temperature, 550
rpm)
(Bronze-Uhle et al., 2017)
Preparation of BSA NPs and BBR-BSA NPs

14. 1st
Centrifuge
at 10,000
rpm for 10
min.
2nd
Centrifuge
at 10,000
rpm for 10
min.
3rd
Centrifuge
at 10,000
rpm for 10
min.
Pellet was
dispersed in 1 ml
of Milli Q water
and sonicated for
5 min.
Pellet was
redispersed in 1
ml of Milli Q
Water and
sonicated for 5
min.
Pellet was
redispersed in 2 ml
of Milli Q water and
stored at -80 ℃ and
next day sample was
lyophilized for
Further
characterization and
biological study
purpose
Purification of BSA NPs and BBR-BSA-NPs

15. OBJECTIVE: 2
CHARACTERIZATION OF BSA NPS AND BBR-BSA NPS

16. The hydrodynamic size distribution of the BSA NPs and BBR-BSA NPs determined by
DLS (Dynamic Light Scattering) was 116 nm and 166 nm respectively.
DLS : Dynamic Light Scattering

17.  The images
clearly shows
that the
nanoparticles
are spherical in
nature.
 The size of the
BSA NPs and
BBR BSA NPs
determined by
FESEM was
104 nm and
110 nm
respectively.
FESEM: Field Emission Scanning Electron Microscopy
BSA NPs
BBRBSA NPs

18. FTIR: FOURIER TRANSFORM INFRARED SPECTROSCOPY
Absorption (cm-1 ) Structural group
3294 cm-1 O-H stretching
1653 cm-1 Amide bond I
1534 cm-1 Amide bond II
Absorption (cm-1 ) Structural group
3479 cm-1 O-H stretching
1643 cm-1 Amide bond I
1515 cm-1 Amide bond II
Absorption (cm-1 ) Structural group
3467 cm-1 O-H stretching
1634 cm-1 -C=N stretching
1505 cm-1 aromatic C=C
vibrations
1227 cm-1 , 1104 cm-1 sulfate ion
Absorption (cm-1 ) Structural group
3293 cm-1 O-H stretching
1648 cm-1 Amide bond I
1528 cm-1 Amide bond II
1233 cm-1 , 1100 cm-1 sulfate ion

19. FTIR confirms the encapsulation of BBR in BSA NPs

20. Thermograms of DSC suggests that BBR encapsulated within BBR
BSA NPs in amorphous form
186 ℃
244 ℃
DSC: Differential Scanning Calorimetry

21. Total weight loss of Pure BSA and Pure BBR observed 75% and
50% respectively. After the loading of BBR, total weight loss of
BBR BSA NPs observed 81% .
TGA: Thermogravimetric analysis

22. 0
200
400
600
800
1000
1200
1400
1 3 5 7
Particlesize(nm)
Time (Days)
pH dependent
pH 5.0
0
50
100
150
200
250
300
350
400
450
500
1 3 5 7
Particlesize(nm)
Time (Days)
Time dependent
Time and pH dependent stability of nanoparticles
As shown in figure, high stability of NPs
was observed at pH 7.4 up to 8 days,
whereas at pH 5.0 the size of NPs was
increased due to aggregation of NPs.
pH 7.4
As shown in Figure, up to 8 days, there
was no significant change in the size of
BBR BSA NPs, which suggests the
stability of prepared nanoparticles in an
aqueous system.

23. OBJECTIVE: 3
ENCAPSULATION EFFICIENCY
AND
IN VITRO RELEASE STUDY OF BERBERINE

24. Entrapment Efficiency, Loading Capacity and % cumulative drug release
Size (nm) PDI EE % LC %
BSA NPs 116.8 0.038 - -
BBR BSA NPs 166.2 0.184 85.65 % 7.78 %
It was observed that
about 97 % of Berberine
was released from
Berberine solution in 2 h
while only 24 % drug
was released from BBR-
BSA-NPs after the same
period of time. then
sustained drug release
observed.
----------------------------------------------
pH 1.2 pH 6.8

25. OBJECTIVE: 4
CYTOTOXIC STUDY OF BERBERINE LOADED BSA
NANOPARTICLES

26. MDA MB 231 cells treated with BSA NPs, Pure BBR and BBR BSA NPs at 25,
50 and 100 µg/ml for 24 h and 48 h. The cytotoxicity was assessed by MTT
Assay. (p < 0.0001 ****)
MTT Assay : MDA MB 231

27. TRYPAN BLUE ASSAY
MDA MB 231 cells treated with BSA NPs, BBR and BBR-BSA NPs at
25 and 50 µg/ml for 24 h and 48 h.

28. 24 h
Control BSA NPs BBR
50 µg/ml
BBR BSA NPs
25 µg/ml
48 h
50 µg/ml
25 µg/ml
APOPTOSIS STUDY
Effects of BSA NPs, BBR and BBR-BSA NPs on apoptotic changes studied
through AO/EtBr staining and visualized by Fluorescence Microscopy.

29. C-6 C-6 BSA-NPs
15 min
30 min
1 h
2 h
4 h
8 h
T im e
FluorescenceIntensity
15
m
in
30
m
in
1
h
2
h
4
h
8
h
0
1.0 10 5
2.0 10 5
3.0 10 5
4.0 10 5
5.0 10 5
C 6
C 6 -B S A -N P s
CELLULAR UPTAKE STUDY
Cellular uptake study of C-6 and C-6 BSA
NPs in MDA MB 231 cells by fluorescence
microscopy. Fluorescent intensity was
quantitatively calculated by ImageJ.

30.  In the present study, we have successfully prepared BSA NPs and berberine loaded BSA
NPs by desolvation method.
 The particles size, morphology and stability analysis indicated that BBR-BSA NPs were
quite stable, spherical in shape and nearly monodisperse in nature.
 The in vitro release profile showed an initial burst release followed by a sustained and
slow release.
 However, cytotoxicity assay (MTT assay), Trypan blue assay and EtBr staining proved
that the prepared NPs are selectively toxic towards breast cancer cells (MDA MB 231) and
kill the cells more efficiently compared to berberine alone.
 Cellular uptake study suggest that BBR BSA NPs could effectively improve the delivery
of BBR into MDA MB 231 cells and therefore it could be responsible for higher
cytotoxicity of BBR-BSA NPs than only BBR against MDA MB 231 cells.
 All the above information suggests that BBR-BSA NPs can be used as a targeted drug
delivery vehicle for intracellular release of berberine for potential therapeutic use in
breast cancer.
CONCLUSION

31. REFERENCES
• Kim, S., Lee, J., You, D., Jeong, Y., Jeon, M., Yu, J., Kim, S. W., Nam, S. J. & Lee, J. E. 2018. Berberine
Suppresses Cell Motility Through Downregulation Of Tgf-β1 In Triple Negative Breast Cancer Cells.
Cellular Physiology And Biochemistry, 45, 795-807
• Remppis, A., Bea, F., Greten, H. J., Buttler, A., Wang, H., Zhou, Q., Preusch, M. R., Enk, R., Ehehalt, R.
& Katus, H. 2010. Rhizoma Coptidis Inhibits Lps-induced MCP-1/CCL2 Production In Murine
Macrophages Via An AP-1 And Nfb-dependent Pathway. Mediators Of Inflammation, 2010.
• Li, Z., Geng, Y.-N., Jiang, J.-D. & Kong, W.-J. 2014. Antioxidant And Anti-inflammatory Activities Of
Berberine In The Treatment Of Diabetes Mellitus. Evidence-based Complementary And Alternative
Medicine, 2014.
• Pan, Y., Zhang, F., Zhao, Y., Shao, D., Zheng, X., Chen, Y., He, K., Li, J. & Chen, L. 2017. Berberine
Enhances Chemosensitivity And Induces Apoptosis Through Dose-orchestrated AMPK Signaling In
Breast Cancer. Journal Of Cancer, 8, 1679.
• Liu, J.-C., Chan, P., Chen, Y.-J., Tomlinson, B., Hong, S.-F. & Cheng, J.-T. 1999. The Antihypertensive
Effect Of The Berberine Derivative 6-protoberberine In Spontaneously Hypertensive Rats.
Pharmacology, 59, 283-289.
• Fan, J., Li, B., Ge, T., Zhang, Z., Lv, J., Zhao, J., Wang, P., Liu, W., Wang, X. & Mlyniec, K. 2017.
Berberine Produces Antidepressant-like Effects In Ovariectomized Mice. Scientific Reports, 7, 1310.

32. • Zhang, Y., Wang, X., Sha, S., Liang, S., Zhao, L., Liu, L., Chai, N., Wang, H. & Wu, K. 2012. Berberine Increases The
Expression Of NHE3 And AQP4 In Sennosidea-induced Diarrhoea Model. Fitoterapia, 83, 1014-1022.
• Zhang, L., Chang, J.-J., Zhang, S.-L., Damu, G. L., Geng, R.-X. & Zhou, C.-H. 2013. Synthesis And Bioactive Evaluation
Of Novel Hybrids Of Metronidazole And Berberine As New Type Of Antimicrobial Agents And Their Transportation
Behavior By Human Serum Albumin. Bioorganic & Medicinal Chemistry, 21, 4158-4169.
• Kulkarni, S. & Dhir, A. 2010. Berberine: A Plant Alkaloid With Therapeutic Potential For Central Nervous System
Disorders. Phytotherapy Research: An International Journal Devoted To Pharmacological And Toxicological Evaluation
Of Natural Product Derivatives, 24, 317-324.
• Zhao, Z., Wei, Q., Hua, W., Liu, Y., Liu, X. & Zhu, Y. 2018. Hepatoprotective Effects Of Berberine On Acetaminophen-
induced Hepatotoxicity In Mice. Biomedicine & Pharmacotherapy, 103, 1319-1326.
• Yu, P., Li, D., Ni, J., Zhao, L., Ding, G., Wang, Z. & Xiao, W. 2018. Predictive QSAR Modeling Study On Berberine
Derivatives With Hypolipidemic Activity. Chemical Biology & Drug Design, 91, 867-873.
• Sun, J., Bao, H., Peng, Y., Zhang, H., Sun, Y., Qi, J., Zhang, H. & Gao, Y. 2018. Improvement Of Intestinal Transport,
Absorption And Anti-diabetic Efficacy Of Berberine By Using Gelucire44/14: In Vitro, In Situ And In Vivo Studies.
International Journal Of Pharmaceutics, 544, 46-54.
• Bashar, A., Hossan, M. S., Jahan, R., Al-nahain, A., Haque, A. M. & Rahmatullah, M. 2014. Berberine: A Potential
Therapeutic Candidate For Breast Cancer. J Pharm Pharm Sci, 3, 1858-1869.
• Bronze-uhle, E., Costa, B., Ximenes, V. & Lisboa-filho, P. 2017. Synthetic Nanoparticles Of Bovine Serum Albumin With
Entrapped Salicylic Acid. Nanotechnology, Science And Applications, 10, 11.
• Kadari, A., Gudem, S., Kulhari, H., Bhandi, M. M., Borkar, R. M., Kolapalli, V. R. M. & Sistla, R. 2017. Enhanced Oral
Bioavailability And Anticancer Efficacy Of Fisetin By Encapsulating As Inclusion Complex With Hpβcd In Polymeric
Nanoparticles. Drug Delivery, 24, 224-232.

33. POSTER PRESENTATION
• Raghu S., K.Patel And S.Patel, “Bovine Serum Albumin Nanoparticle For
The Efficient Delivery Of Anticancer Drug (Berberine): Preparation,
Characterization And In Vitro Studies” 6th World Congress On Nanomedical
Sciences At Vigyan Bhawan, New Delhi From December 9-14, 2018. ISNSCON
2018.
• Raghu S., K.Patel And S.Patel “Enhanced Oral Bioavailability And
Anticancer Efficacy Of Berberine By Encapsulating In BSA Nanoparticles”
International Conference On Molecular Basis Of Diseases And Therapeutics At
Central University Of Rajasthan, Ajmer, Rajsthan From March 8-10, 2019.
ICMBDT 2019.

34. ACKNOWLEDGEMENT
• I Would Like To Express My Special Thanks To:
 Dr. Sunita Patel, Assistant Professor, SLS, CUG
 Prof. S.A.Bari, VC, CUG
 Prof. J.P.N.Mishra, Dean, SLS, CUG
 All Faculties Of SLS
 CIF, CUG
 IIT Gandhinagar
 UGC-NFSC
 Central University Of Gujarat, Gandhinagar
 My Classmates, Seniors And Friends
 My Parents