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

01/05/2023 5

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

01/05/2023 6

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).

.

01/05/2023 7

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

01/05/2023 9

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

01/05/2023 10

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

01/05/2023 11

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)

01/05/2023 12

01/05/2023 13

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 !!!