Formulation and in vitro evaluation of quercetin loaded carbon nanotubes for ...
Wei Lu & Fiona 25 Feb 2015 FYP presentation! NP
1. Cultivation and Screening of
Microalgae Isolates for Anti-Tumour
Bioactive Compounds
Chin Wei Lu
Lim Wei Xin Fiona
SUPERVISOR: Dr. New Jen Yan
CO-SUPERVISOR: Dr. Charmaine Lloyd
2. Microalgae
Microalgae are photoautotrophic, unicellular algae cells
Being explored as alternative sources, over terrestrial
plants, of high-value products, such as renewable
biofuels and nutritional supplements (e.g. dietary anti-
oxidants, vitamins)
Great diversity
Simple growth requirements
High growth efficiencies
Mass cultivation offshore
Introduction
Lugol’s iodine-stained microalgae isolated
from local water bodies
1
3. Nitrogen-deficient cultivation increased lipid content in
Chlorella vulgaris microalgae from 14.5% to 24.6% of dry
weight (Mujtaba et al., 2012)
Ultraviolet-A irradiation increased both lipid content and
degree of unsaturation, in Nitzschia closterium
(Bacillariophyceae) and Isochrysis zhangjiangensis
(Chrysophyceae) microalgae (Huang and Cheung., 2011)
Introduction
2
Flexibility to alter biomass
composition
4. Introduction
3
Anti-cancer activity in
macroalgae
Reduction in tumour size of Agrobacterium
tumefaciens-infected potato discs, after
treatment with ethanol crude extracts from
Jania rubens algae (Ibrahim et al., 2005)
Jania rubens red algae
Pepsin-digested extracts from Caulerpa
microphysa algae induced tumour
shrinkage in immunocompromised mice
transplanted with human leukaemia cell
lines (Lin et al., 2012)
Caulerpa microphysa algae
http://www.umema.it/Alg
he/album/Rosse/slides/02
%20Jania%20Rubens.html
http://biogeodb.stri.si.edu/pacificalgae/specie/19
5. I. To determine if crude extracts from local water bodies-
originated microalgae species (vs38, vs88, vs31 and KK6)
have anti-tumour effects against cancerous basophils
KU812 and oestrogen receptor-negative breast cancer cell
line MB231
II. To compare the efficacy of these crude extracts with anti-
cancer antibiotic Actinomycin-D in inhibiting KU812 and
MB231 proliferation
Objectives
4
6. Cultivation of microalgae strains
• 4 morphologically-distinct
microalgae strains isolated from
local water bodies
• Incubated in an enclosed room
at (25±1)⁰C with illumination
• Half of the respective algal
cultures were irradiated under
UV-C for 4 hours (with swirling
every 10 minutes) -> 24 hours
recovery time
vs38
KK6
vs88
vs31
Materials and Methods
5
7. Harvesting of microalgae
strains
Washing -> Lyophilisation -> Solvent extraction
(Hexane & Ethanol & dH2O)-> Ultra-sonication ->
Vortex with micro glass beads -> Incubation ->
Evaporate to dryness
Mass of dried extracts were recorded
Re-dissolve dried extracts in DMSO to achieve a
Master stock of microalgae extract(2000mg/mL)
Materials and Methods
Microtube
Glass tube
2000mg/mL
6
8. Anti-tumour test for microalgae crude
extracts and Actinomycin-D
1. Cancer cells were seeded
at a density of 2.0x105 living
cells/mL in each well on a
96-well plate, in replicates
of five for each working
concentration
2. Cells then incubated at
37◦C, 5% CO2 for 24 hours
to acclimatise/adhere to
substratum
3. Cells then added with
microalgae crude extract
master stocks, to the following
working concentrations:
o 2.0mg/mL
o 1.0mg/mL
o 0.5mg/mL
o 0.25mg/mL
o 0.0625mg/mL
Materials and Methods
7
9. 4. Treated cells then
incubated at 37◦C,
5% CO2 for 48 hours
5. Cell viability at each
microalgae crude extract
working concentration
assessed using 3-(4,5-
dimethylthiazol-2-yl)-2,5-
diphenyltetrazolium
bromide (MTT) Assay
Anti-tumour test for microalgae crude
extracts and Actinomycin-D
Statistical analysis
Significant differences in cell viability between samples
were tested for using Student’s t-test for non-paired
samples and Mann-Whitney-U test
Materials and Methods
8
10. Negative control
Cancer cells cultured in
pure culture medium only
Safe DMSO control
Cancer cells cultured in
0.1% (v/v) DMSO
MTT control
Cancer cells cultured in
50% (v/v) DMSO
Materials and Methods
9
11. Analysis of results
Growth inhibitory or stimulatory effects of microalgae crude
extracts assessed only at 0.25mg/mL working concentration
Growth stimulatory effect population of living cancer cells in
microalgae test is significantly larger than in negative control
Growth inhibitory effect population of living cancer cells in
microalgae test is significantly smaller than in negative control
Materials and Methods
10
12. Growth inhibition efficiency =
[(cell viability in negative control – cell viability in microalgae test)/cell viability in negative control] x 100%
MB231 cell viability in stressed and non-stressed microalgae
vs88 distilled water crude extract tests, and in their
respective negative controls, after 48-hour exposure
(representative bar chart)
Materials and Methods
11
Viable breast cancer MB231 cells (after MTT assay) seen
under microscope. LEFT: incubation with inhibitory
microalgae crude extract. RIGHT: negative control
13. Results and Discussion
Effects of test
microalga on
cancerous
basophils KU812,
BY STRAIN
Y-axis = Population of living cancer cells present
Blue – test microalgae crude extract
Pale blue – negative control
12
14. Results and Discussion
Effects of test
microalga on
breast cancer
cell line MB231,
BY STRAIN
Dark grey – test microalgae crude extract
Pale grey – negative control
Y-axis = Population of living cancer cells present
13
15. Growth of cancerous basophils KU812 is significantly inhibited by
microalgae strain vs31, but not by vs38, vs88 and KK6.
Growth of breast cancer cell line MB231 is significantly inhibited
by microalgae strains vs88 and vs38, but not by vs31 and KK6.
1) Microalgae strains vs31 and vs88 are promising candidates for
further exploration into their anti-tumour potentials against
cancerous basophils and breast cancer respectively
Possible implications
Results and Discussion
14
16. Possible implications
2) Modes of action of anti-tumour bioactive compounds from vs31,
vs38 and vs88 possibly specific and targeted
Since proliferation of cancerous basophils KU812 and breast
cancer cells MB231 each inhibited by different microalgae strains
Interference only with oncogenic events pertaining to respective
cancer cell line, not general proliferative mechanisms (e.g.
disruption of DNA replication) (National Cancer Institute, 2014)
Results and Discussion
15
17. Test microalga with growth inhibitory effects,
BY EXTRACTION SOLVENT
Hexane crude extract Ethanol crude extract Distilled water crude
extract
Results and Discussion
16
18. Possible implications
3) Anti-tumour bioactive compounds from vs38, vs88 and vs31 are
likely of medium to high chemical polarity
12 out of 15 microalgae crude extracts inhibiting cancer growth
were isolated using polar ethanol and distilled water extraction
solvents
Extraction solvents “capture” compounds of like polarity
Results and Discussion
17
19. 37.1% 13.4%
30.4% 20.8% 34.2% 18.9%
Results and Discussion
Effects of test
microalga on
cancerous basophils
KU812, BY GROWTH
CONDITIONS
18
Non-stressed Stressed
Non-stressed Stressed StressedNon-stressed
21. Possible implications
4) Bioactive compounds inhibiting KU812 and MB231 cancer
proliferation from vs31, vs38 and vs88 likely NOT lipids in nature
Non-stressed crude extracts should theoretically comprise
smaller lipid content than stressed counterparts, due to lack of
ultraviolet-C irradiation
Greater proportion of non-lipids stronger growth inhibition
efficiencies
Results and Discussion
20
22. Nature of microalgae crude extracts (inhibits or stimulates cancer cell growth) on KU812 cell line (purple)
and MB231 cell line (blue)
Results and Discussion
Test microalga with growth stimulatory effects,
BY GROWTH CONDITIONS
21
23. 5) Lipids may play an assistive role in cancer cell proliferation
Crude extracts from stressed microalgae should theoretically
comprise greater lipid content from ultraviolet-C irradiation
Greater abundance of lipids in stressed crude extracts translated to
poorer cancer growth inhibition
Findings corroborate other literature reporting growth-stimulating
effects of lipids on cancerous tumours (Baenke et al., 2013, Huang and Cheung., 2011)
Signalling molecules for cancer cell proliferation, migration,
angiogenesis
Raw material for membrane synthesis and energy generation
Unsaturated lipids increase cell membrane permeability and
nutrient intake
Possible implications
Results and Discussion
22
24. Microscopic observation
Figure 3.9
Anti-tumour effect determined at 0.25mg/mL extract exposure -> could be clearly
observed using microscopy at 2mg/mL extract exposure
Results and Discussion
2mg/mL vs31
stressed Ethanol
2mg/mL KK6
stressed Ethanol
Complete
medium only
0.1% (v/v) DMSO
only [safe dose]
KU812
2mg/mL vs88
stressed dH2O
2mg/mL KK6
stressed dH2O
Complete
medium only
0.1% (v/v) DMSO
only [safe dose]
MB231
23
25. Anti-tumour test with Actinomycin-D
The smaller the IC50 values, the greater the potency
of a particular compound or drug.
Gentler gradient= Wider therapeutic
window=adverse drug event unlikely to be
observed with subtle changes in drug
concentration.
Steeper gradient= Narrower therapeutic windows
adverse drug event likely to occur with subtle
changes in drug concentration.
Results and Discussion
24
26. Anti-tumour test with Actinomycin-D
Figure
3.11
MB231 exposed
to..
Gradient IC50 value
Stressed vs31 dH2O
crude extract
-3.72 %.mg-1.mL 9.38 x 1010 mg/mL
Actinomycin-D -20.8 %.mg-1.mL 3.81mg/mL
Wider therapeutic window, Less potent
Results and Discussion
25
30. Anti-tumour test with Actinomycin-D
Non-stressed
vs31 KU812
dH2O
KU812
exposed to..
Gradient IC50 value
Non-stressed
vs31 dH2O
crude
extract
+16.9 %.mg-
1.mL
3.35 x 10-
2mg/mL
Actinomycin
-D
-14.8 %.mg-
1.mL
13.9mg/mL
Figure
3.12
• At 0.25mg/mL of the
extract significantly
depicted that it
inhibited the
proliferation of KU812
• However, a positive
correlation observed
between cell viability
& concentration of
the extract
• High amount of
bioactive compounds
which had promoted
cellular proliferation
Results and Discussion
-D
29
31. Anti-tumour test with Actinomycin-D
-D
-D
-D
-D
-D
-D
Non-stressed
vs31 KU812
Hexane
Stressed vs31
KU812
Hexane
Non-stressed
vs31 KU812
Ethanol
Non-stressed
vs31 KU812
dH2O
Stressed vs31
KU812
Ethanol
Stressed vs31
KU812 dH2O
Figure
3.12
• 50% tested microalgae extracts on KU812 cell line (B, C and
F) displayed higher IC50 values than Actinomycin-D
Crude
Concentration of anti-tumour metabolites smaller per
unit volume
• Other 50% of the tested microalgae extracts on KU812 cell
line (A, D and E) revealed smaller IC50 values than
Actinomycin-D Crude
High concentration of anti-tumour metabolites
OR
Small concentration of anti-tumour metabolites had
targeted a critical apoptotic cascade-> greater effect in
inhibiting KU812 growth
Results and Discussion 30
32. • Identity of components exhibiting growth
inhibitory effects could have been conclusively
verified using chromatography
• Prospective use of microalgae crude extracts as
anti-tumour therapy in humans could have been
substantiated by conducting cytotoxicity tests on
non-transformed human cell lines
• Examining local microalgae species for anti-
oxidant properties and potential to repress
malignant transformation and cancer onset
Future Work
31
33. Summary
32
Investigating anti-tumour
effects from microalgae
isolated from Singapore’s
water bodies
KK6
(NIL)
vs31 inhibited basophil
leukemic cells
vs38 & vs88
inhibited breast
cancer cells
12/15 are ethanol
& dH2O
Anti-tumour bioactive compounds
= medium to high chemical
polarity, with targeted modes of
action
3/15 are hexane
Lipids
Stressed
Non-
stressed
Lipids
Side
Project
Varying potencies & therapeutic
windows as compared to those of
Actinomycin-D
35. Baenke, F., Peck, B., Miess, H., and Schulze, A., 2013. Hooked on fat: the role of lipid synthesis in cancer
metabolism and tumour development. Disease models and mechanisms, 6(6), pp. 1353-1363.
Boopathy, N. S., and Kathiresan, K., 2010. Anticancer Drugs from Marine Flora: An Overview. Journal of
Oncology, Volume 2010, pp. 1-18.
Huang, J.-h. J., and Cheung, C.-K. P., 2011. +UVA treatment increases the degree of unsaturation in
microalgal fatty acids and total carotenoid content in Nitzchia closterium (Bacillariophyceae) ad Isochrysis
zhangjiangensis (Chrysophyceae). Food Chemistry, Volume 129, pp. 783-791.
Ibrahim, A. M. M., Mostafa, M. H., El-Masry, M. H. and El-Naggar, M. M. A., 2005. Active biological materials
inhibiting tumour initiation extracted from marine algae. Egyptian Journal of Aquatic Research, Volume
31(1). pp. 146-155.
Lin, H. C., Chou, A. T., Chuang, M. Y., Liao, T. Y., Tsai, W. S., and Chiu, T. H., 2012. The effects of Caulerpa
microphysa enzyme-digested extracts on ACE-inhibitory activity and in vitro anti-tumour properties. Food
Chemistry, Volume 134. pp. 2235-2241.
Mujtaba, G., Choi, W., Lee, C.-G., and Lee, K., 2012. Lipid production by Chlorella vulgaris after a shift from
nutrient-rich to nitrogen starvation conditions. Bioresource Technology, Volume 123, pp. 279-283.
National Cancer Institute, 2014. Targeted Cancer Therapies. [Online]
Available at: http://www.cancer.gov/cancertopics/factsheet/Therapy/targeted
[Accessed 12 December 2014].
Singh, S., Kate, B. N., and Banerjee, U. C., 2005. Bioactive Compounds from Cyanobacteria and
microalgae: An Overview. Critical Reviews in Biotechnology, Volume 25, pp. 73–95
Bibliography
34
36. Cultivation & harvesting of
microalgae strains
Mass of dried extracts were recorded
Re-dissolve dried extracts in DMSO to achieve a
Master stock (2000mg/mL) before serial dilutions to
produce 4 more Master stocks:
2000mg/
mL
1000mg/
mL
500mg/mL 250mg/mL 62.5mg/mL
Materials and Methods
Microtube
Glass tube
35
37. Microscopic observation
KU812
Comparison
MB231
Comparison
A B
G H
C D E
I J K
Figure 3.9
• Cells were viewed under 400x
magnification using Olympus
CK40 inverted microscopy
• Comparison of the cell
morphology/cell confluency &
presence of dark blue
precipitate inside cells after
incubation with MTT for~3hours
• C and I are negative controls
(exposed to complete
medium only)
• D and J are “Safe-DMSO”
controls
• E and K are “Lethal-DMSO”
controls
Effectiveness of
microalgae extracts on
cancer cells?
Cell type Effective Ineffective
KU812 A B
2mg/mL
vs31
stressed
Ethanol
2mg/mL
KK6
stressed
Ethanol
MB231 G H
2mg/mL
vs31
stressed
dH2O
2mg/mL
KK6
stressed
dH2O
• Anti-tumour effect determined
at 0.25mg/mL extract
exposure -> could be clearly
observed using microscopy at
2mg/mL extract exposure
Results and Discussion 36
38. Microalgae strains vs31, vs38 and vs88 displayed anti-cancer
properties, except for KK6
Anti- tumour bioactive compounds likely of medium to high
chemical polarity, with targeted modes of action
Anti-tumour bioactive compounds unlikely to be lipids-based
The peculiar case of non-stressed vs31 distilled water against KU812
(Figure 3.12E)
Varying potencies & therapeutic windows as compared to those of
Actinomycin-D
Possible clinical treatment of cancer with local microalgae strains in
future
Conclusions
37
Editor's Notes
Piqued the interest of not only scientific medical communities but also of commercial companies due to… and anti-microbials (anti-biotics)
Microalgae are self-sufficient units
Reduction in tumour size of Agrobacterium tumefaciens-infected potato discs, after treatment with ethanol crude extracts from Jania rubens algae
Extracts from Caulerpa macrophysa algae induced tumour shrinkage in immunocompromised mice transplanted with human leukaemia cell lines (Lin et al. 2012)
Alkaloids, diacylglycerols, polyphenols and sulfated polysaccharides in nature (Boopathy and Kathiresan 2010; Singh et al. 2005)
In MM talk about how growth inhibition efficiency is calculated…give a brief outline: basically cancer cells were incubated for 48 hours with differing working concentrations of microalgae crude extracts
96 well microtitre plate
LOOK UP OTHER CELL VIABILITY ASSAYS AND COMPARE ACCURACY
Types of controls used and how we assessed properties at 0.25mg/mL = cell viability should be significantly smaller in test than negative control and growth inhibtion efficiency assessed using…
Criteria for verification of growth inhibitory effects is explained under MM (cell viability in test wells should be sig smaller than in negative controls)
By 13.4 percent to 37.1% of the negative controls
CHANGE 30% and 47.5%!!!
And Not general proliferative mechanisms (e.g. DNA cross-linking, blocking nucleotide synthesis, etc.)
Similar to gleevec interfering only with cases of ALL triggered by constitutive activation of tyrosine kinases…
Reiterates fact that anti-tumour compounds likely not lipids in nature
Show stronger growth inhibitory effects against ku812 than crude extracts from stressed
Paired analysis. Repeat rationale for stressing microalgae
Tended to show a larger difference in cell viability from negative control than stressed
Crude extracts from non-stressed vs31 tended to show stronger growth inhibition efficiencies than their stressed counterparts (1.5 to 3 folds stronger)
Growth inhibition efficiencies of non-stressed vs38 and vs88 crude extracts against MB231 generally higher than stressed counterparts, also consistently above 30.0%
Growth inhibition efficiencies of stressed vs38 and vs88 crude extracts against MB231 were more fluctuated (from 24.0% to 46.8%)
Indicates that whatever compounds inhibiting tumour growth not likely to be lipids, and also lipids might pay an assistive role in cancer proliferation. Next slide, how our findings back others’ work about lipids promoting cancer growth. Also by how x out of y extracts promoting cancer were from stressed and x out of y extracts inhibiting cancer growth were ethanol distilled water and so likely polar
Four out of five microalgae crude extracts promoting cancer cell growth derived from stressed microalgae
Mostly due to dysfunctional lipid metabolism and regulation in other cancers….beta-oxidation of lipids to produce energy?
At 0.25mg/mL
Components of microalgae crude extracts to be tested individually for growth inhibitory effects against cancer cell lines
Oxidative stress implicated in oncogenesis
Anti-oxidant properties discovered in various algae species (e.g. Himanthalia elongata)
Anti-tuomour bioactive compounds unlikely to be lipid-based -> based on results derived from hexane extracts and harvested UV-C irradiated microalgae
Anti-tuomour bioactive compounds unlikely to be lipid-based -> based on results derived from hexane extracts and harvested UV-C irradiated microalgae
