BIOMEDICAL NANOTECHNOLOGY

LECTURE 19: in vitro METHODS TO STUDY ANTIBACTERIAL AND ANTICANCER

PROPERTIES OF NANOMATERIALS

Dr.P.GOPINATH
DEPARTMENT OF BIOTECHNOLOGY

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 Contents

Various in vitro methods to study

antibacterial and anticancer
properties of nanomaterials

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Methods to study Antibacterial
properties of nanomaterials

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 Turbidity assay
(a)

S. aureus
MTCC 737

Treated with different

concentrations of CZ
(b) nanofibers

GFP E. coli

RSC Advances, 2016, 6, 115021-115028 4

Determination of MIC and MBC of Ag-ZnO Nanocomposite
by Optical density measurements

Effect of different concentrations of Ag-ZnO core-shell nanocomposite [Zn:Ag(1:0.88)]

on growth of (A) S.aureus (n=3) and (B) recombinant GFP E.coli (n=3).
From Visual turbidity Analysis and Optical density measurements
60µg/mL and 70µg/mL was found to be the minimum inhibitory concentration (MIC)
and minimum bactericidal concentration (MBC) value against S.aureus
550 µg/mL and 600 µg/mL were estimated as MIC and MBC value against GFP E.coli

Colloids and Surfaces B: Biointerfaces 2014,115, 359–367. 5

Effect of various concentration of AgNPs

Langmuir, 2006, 22, 9322-9328.

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Fluorescence micrograph of GFP E. coli
A B

μ μ

Control Ag NP treated

Langmuir, 2006, 22, 9322-9328.

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Time dependent fluorescence micrographs

Langmuir, 2006, 22, 9322-9328.

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 Fluorescence spectrophotometric and microscopic analysis

a) No fluorescence was observed in GFP E. coli treated with 10 or 12 mg of composite

nanofibers; this suggested complete eradication of bacteria at these concentrations
b) Effects of different concentrations of nanofibers on the viability of antibiotic-resistant GFP
E. coli: (A) PEO–PCL nanofiber-treated cells alone and (B–D) composite nanofiber-treated
cells of various weights (6, 8, and 10 mg).
Journal of Applied Polymer Science, 2015, 132, 42473. 9
Antibacterial assay with the disc diffusion method

Disc diffusion assay a

showed (a) control b
nanofiber b) 1wt.% Ag
(c) 2wt.% Ag (d) and
3wt. % Ag
d
incorporated
composite nanofiber
c

Journal of Applied Polymer Science, 2015, 132, 42473. 10

Colony counting method

Journal of Applied Polymer Science, 2015, 132, 42473. 11

Bacterial colony count

Langmuir, 2006, 22, 9322-9328.

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Morphological observation of the treated bacterial cells by SEM

FE SEM micrograph showed control nanofiber treated GFP E. coli bacteria (A-C)
and GFP E. coli treated with composite nanofibers; this suggested complete cell
death of bacteria at these concentrations.
Journal of Applied Polymer Science, 2015, 132, 42473. 13
Transmission electron microscopic (TEM) Images
A

A, a single bacterial cell

B, expanded view
indicating the
presence of AgNPs
on the cell membrane
Langmuir, 2006, 22, 9322-9328.

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 Antibacterial mechanism

Schematic representation of antibacterial mechanism of core-shell Ag-ZnO nanocomposites

Colloids and Surfaces B: Biointerfaces 2014,115, 359–367. 15

Methods to study Anticancer properties
of nanomaterials

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 Apoptosis and necrosis
• Apoptosis (“normal” or “programmed” cell death)
is the physiological process.

• Necrosis (“accidental” cell death) is the

pathological process.

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 Apoptosis and necrosis
Apoptosis
• Membrane blebbing, but no loss of Necrosis
integrity • Loss of membrane integrity

• Begins with shrinking of cytoplasm & • Begins with swelling of cytoplasm &
condensation of nucleus mitochondria

• Ends with fragmentation of cells into • Ends with total lyses

smaller bodies
• Random digestion of DNA (smear of
• Non-random mono- and DNA after agarose gel electrophoresis)
oligonucleosomal length fragmentation
of DNA (Ladder pattern after agarose gel
electrophoresis)
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Apoptosis and necrosis

Source: Andreas Gewies (2003), ApoReview - Introduction to Apoptosis, 1-26. 19

 Detection of apoptotic cells
• Surface morphology

• Cell membrane integrity

• Mitochondrial function

• Nuclear events

• DNA cleavage

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Dye based cell viability assay

Trypan blue staining of dead cells

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MTT assay/ Cell viability assay

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Cell viability assay

Nanotechnology , 2008, 19 (7). 23

 Cell viability assay
A549 cells MCF-7 cells

IC50 -5 µM IC50 -2.6 µM

Bare niclosamide (in water) showed a nontoxic effect due to its practical insoluble nature in
aqueous medium
RSC Advances, 2015,5,12078–12086 24
Acridine orange/ ethidium bromide (AO/EB) staining
• Fluorescent DNA
intercalating dye.
• permeable to all cell
nuclei and emit green
fluorescence.

• Fluorescent DNA
intercalating dye.
• Enter only the membrane
compromised nucleus to emit
orange-red fluorescence

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 AO/EB staining
Acridine Orange (AO) BHK21 Control BHK21 treated
Ethidium Bromide (EB)
Live

EA-Early Apoptosis

LA-Late Apoptosis

Necrosis
Baby Hamster Kidney (BHK21) cells
Human colorectal adenocarcinoma (HT29) cells HT29 Control HT29 treated
Molecular Biotechnology , 2008, 39, 39-48. 26
Hoechst 33342 / Rhodamine B (Hoechst-rho B) staining

Hoechst 33342
• membrane permeable
nuclear staining dye that
emits blue fluorescence
Rhodamine-B
• membrane permeable
when bound to dsDNA.
dye that stains the
mitochondria and
• Use to differentiate
cytoplasmic
condensed pycnotic nuclei
compartments.
(nuclei with condensed
chromatin) from normal
nuclei.
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 Hoechst-rho B staining

White arrows indicate chromatin condensation (dark spots) and yellow arrows point towards
cytoskeleton compaction.

RSC Advances, 2015,5,86242-86253 28

 Morphological examination by SEM

• Untreated cells- spindle-shaped, well-attached to the surface and intact membrane

morphology.
• IC50 treated cells- shrunk in size, rounded in shape, loosely attached and exhibited
membrane blebbing which are the hallmarks of apoptotic cell death.

Journal of Materials Chemistry B, 2015,3,1208–1220 29

Atomic force microscopic (AFM) images

A B

LA EA

BHK21 control BHK21 treated

Molecular and Cellular Biochemistry,2009, 324, 21-29. 30

 Atomic force microscopic (AFM) images

BHK21 control

BHK21 treated

Molecular and Cellular Biochemistry,2009, 324, 21-29. 31

 Cellular DNA fragmentation ELISA

Source: Roche Applied science catalogue

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 Cellular DNA fragmentation ELISA

Nos. 1: untreated controls, 2: 11.0 μg/mL of Ag NPs; 3: 20 mM 5-FU, 4: 11.0 μg/mL of

Ag NPs with 20 mM 5-FU; 5: 20 mM 5-FU on UPRT transduced cells; 6: combine
treatment of 11.0 μg/mL Ag NPs with 20 mM 5-FU on UPRT transduced cells.

Nanotechnology , 2008, 19 (7). 33

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 Apoptotic DNA laddering

Source: Roche Applied science catalogue

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 MTT, ELISA and DNA Laddering
CLSM

MTS

DNA laddering

ELISA

•Caspase-3 activates CAD (Caspase-activated DNase) leads to cleavage of chromosomal DNA.

•The oligonucleosomal DNA fragments results in a distinct laddering pattern.

Molecular and Cellular Biochemistry,2009, 324, 21-29. 35

Fluorescence-activated cell sorting (FACS)

http://www.biomedicinapadrao.com.br/2014/07/principais-metodos-utilizados-em.html
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 Flow cytometric analysis

Flow cytometry measures light scattering properties of cells (correlate to cell

sizes and cell granularities) and fluorescence emissions of molecules
attached to cells (usually conjugates of fluorochrome and antibodies to cell
surface proteins).

http://www.bioinformin.net/cytometry.php 37
Flow cytometric analysis

Necrotic Late apoptotic

cells cells

Alive Early apoptotic

cells cells

Colloids and Surfaces B: Biointerfaces, 2010 Jun 1;77(2):240-5. 38

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ROS assay

(DCFH-DA )
2’,7’-dichlorfluorescein
diacetate

(DCFH)
2’,7’-dichlorfluorescein

(DCF)
dichlorofluorescein
 Determination of ROS by 2’,7’-dichlorofluorescin diacetate
(DCFH-DA) Assay
ROS levels can be quantified by Non- Fluorescent DCFH-DA
determining the percentage of
fluorescent cells using a flow
cytometer. Passive
Cellular
uptake
Esterase
DCFH (λex = 495 nm; λem = 529 nm)
Non- Fluorescent DCFH
Gated population of Cells:

• R2- Non fluorescent cells. ROS

• R3- Fluorescent cells (ROS

Fluorescent DCF
production).

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 ROS assay
Untreated 0.5x IC50 IC50 2x IC50

MCF-7

A549

Flow cytometric analyses of ROS production in A549 and MCF-7 cells treated with
different concentrations of FA-BSA-Ag NPs.

RSC Advances, 2015,5,86242-86253 41

 ROS estimation by DCFH-DA

• The FA functionalized Fe3O4 NPs catalytically generate ROS at its surface and exerts oxidative stress on
KB cells in the vicinity which is further augumented in the presence of AMF.
• With increase in treatment time point, percentage of cells with higher ROS subsequently increased as
more and more Fe3O4 NPs are released from the composite nanofibers and taken up by cells.

RSC Advances, 2016,6,46186-46201 42

Analysis of Fluctuation in Mitochondrial Membrane Potential
(MMP) by Rhodamine 123 Staining
• Change in the mitochondrial membrane
permeability with a loss in MMP (ΔΨ) is an
indicator of early apoptotic events.
• Rhodamine 123: a cationic dye can rapidly
diffuse inside the mitochondrial interior and
can reflect the changes in MMP.
• a1-c1: No loss in red fluorescence, or no loss
of ΔΨ ~ untreated cells.
• d1-e1: Significant decline in rhodamine
fluorescence, or loss of ΔΨ.
• The results reveal a remarkable decline in
MMP confirming induction of apoptosis in
MCF-7 cells after My-g-G5/TAM treatment.

RSC Advances, 2016, 6,24808-24819 43

 Gene expression analysis by RT-PCR
Pro-apoptotic genes
Anti-apoptotic genes

(A) Semi-quantitative RT-PCR analysis of apoptotic signalling genes. Lane 1 and 2: untreated
and CD-Ag@ZnO NC treated MCF-7 cells (50 µg mL-1) respectively. (B) Fold difference in
gene expression in treated MCF-7 cells as compared to untreated MCF-7 cells.
Journal of Materials Chemistry B, 2015,3,1208–1220 44
Schematic illustration of events

• Upon cellular uptake, the NC

induced oxidative stress by
evoking ROS and triggered
the p53-mediated apoptotic
pathway.

• Cascade of events lead to

changes in mitochondrial
membrane permeability,
resulting in up-regulation of
caspase 3 (final executioner
of apoptosis).

Journal of Materials Chemistry B, 2015,3,1208–1220 45

 Summary

Various in vitro methods to study

antibacterial and anticancer
properties of nanomaterials

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