dr mudasir ahmad lone - nanotalk
TRANSCRIPT
Dr. Mudasir A Lone
Nanotechnology Based Methods For The Screening Of Medically
Important Molecules And Cells
By
IgG Antibody Molecule
(~ 12 -15 nm)
Platelet
width (~ 10−1000 nm) intercellular (~
1.1nm)
Antibody and Platelet
1. Belong to class of medicines known as protein therapeutics
2. Effective in treating some forms of cancers and tumours
3. Abnormal behaviour has adverse effects
Significance of IgG Antibodies
and Platelets
1. Normal functioning/clumping of platelets produces clots in order to
prevent bleeding.
2. However, their abnormal behaviour could lead to strokes in heart/brain
Molecular and Cellular transformation
Influencing Factors
Unstable Antibody MoleculeStable Functional Antibody Molecule
Normal CellAbnormal Activated Cells
- Change in shape
cInfluencing Factors
c
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I. The clumping together of two or more than two activated platelets.
II
Abnormal Activated Cells
Clumping
Cellular aggregate / clot
Unstable Antibody Molecules
Clumping
Aggregated Antibody Molecules
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I. Conventional Microscopy:
a. Overlooks imaging single molecules or small molecular aggregates
with nano-dimensions
b. There is no emphasis on which cells are more prone to aggregation and
why?
c. Lacks the ability to measure the strength of the inter-molecular/inter-cellular
interaction forces that lead to aggregation.
II. Where is the room to improve our understanding?
a. Identification of morphologically different molecules and platelets (activated)
that participate in aggregation
b. Measurement and strength of the inter-molecular and inter-platelet interaction
forces that exist between different molecules/morphologically different platelets
(activated).
.
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I. Imaging of Molecules and Different Cell
Types With Atomic Force Microscopy.
Instead of using an incident beam to visualize
a sample, as would be the case in classical
Microscopy, AFM senses the small forces
(in the piconewton range, ,10-12
N) that act
on the sample surface.
a. Molecules and Cells have been imaged with
AFM.
b. Can provide high-resolution images of
molecules cell surfaces under physiological
conditions.
AFM Study of the Effect of Multiple freeze drying on IgG
- AFM images reveal both crystalline and amorphous features. Globular, protein like features can also be observed.
- The size of the smaller globular features is similar to the size of monomeric IgG (~15-20nm) reported in previous AFM studies
(Ultramicroscopy 105:103-110). The larger globular features likely represent aggregates of IgG
250nm 250nm 250nm
Cycle 1 Cycle 2 Cycle 3
AFM images of freeze dried IgG samples, prepared without excipients (starting concentration 2mg/ml in 0.01M PBS)
Globular features
Crystalline
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AFM study of IgG Freeze Dried with 20mM Mannitol
- AFM images show both amorphous and crystalline features.
- Globular features appear to be associated with distinct regions on the sample
surface e.g. some crystalline features do not appear to be coated with a protein like
layer.
250nm250nm 250nm
A B C
150 nm
D
Crystalline
Amorhous
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IgG Freeze Dried with Sucrose and Mannitol in Combination [1]
- Images of once freeze dried IgG with Sucrose (20mM) and Mannitol (40mM) in
combination
- An increase in the molar concentration of mannitol reveals distinct crystalline
features in some areas.
- The globular features of IgG profoundly appear to be associated with the amorphous
material.
250nm 250nm 250nm
A B C
150nm
D
Crystalline
Amorhous
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This image, taken with atomic force
microscopy, shows E. coli (2-6µm) bacteria
after they have been exposed to the
antimicrobial peptide CM15. The peptides
have begun destroying the bacteria’s cell
walls.
(http://web.mit.edu/newsoffice/2010/micropeptides-
0315.html)
Topographical rearrangements shown by AFM images of myoblasts fusing
due to cytoskeletal dynamics during myogenesis.
(http://www.mechano-biology.ethz.ch)
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I. Characterization of Activated Cells On The Basis of Their Binding Strength With Bio-membrane Force Probe (BFP).
A B C D
Aggregation receptor is glycoprotein IIb/IIIa (gpIIb/IIIa); calcium-dependent for
fibrinogen. Other receptors areGPIb-V-IX complex (vWF) and GPVI (collagen)
Connecting agent such as fibrinogen, fibronectin, vitronectin, thrombospondin, and
vWF (von Willebrand factor)
A
(a) Specific, (b) non-specific, (c) no adhesion (d) multiple force
displacement curves of the interaction
Plot of adhesion force or binding interaction
strength.
I. Determination of molecular/cellular size and identification of different cell types and those more
prone to aggregation is possible with AFM imaging.
II. Measurement of ligand (fibronectin/fibrinogen) – cell surface receptor interactions would be ideal to
determine the protein-protein interactions
III. Strength of platelet interactions that contribute maximum to the aggregation is possible with AFM,
and hence, would be useful for the quantification of cells showing high propensity to aggregate.
Thanks For Your Patience And Kind Attention Attention !!!