Master’s Seminar On ATOMIC FORCE

MISCROSCOPY IN DRUG DISCOVERY

Presented ByChandan PatilI MVScV-1661/16

Department Of Veterinary Pharmacology & Toxicology DUVASU ,Mathura

Types of microscopy

HistoryThe precursor to the AFM, the Scanning

Tunneling Microscope (STM), was developed by Gerd Benning and Heinrich Rohrer in the early 1980s at IBM Research –Zurich, a development that earned them

the Nobel Prize for Physics in 1986.

Binnig invented the Atomic-Force Microscope and the first experimental

implementation was made by Binnig,Quate and Gerber in 1986.

Salient Features Very-high-resolution type of Scanning Probe Microscopy (SPM), with demonstrated resolution on the order of fractions of a nanometer.

The information is gathered by "feeling" or "touching" the surface with a mechanical probe.AFM permits the three dimensional(3D) imaging of membranes and biomolecules with molecular and sub-molecular resolution.

AFM sample preparation is fairly simple and quick.

AFM does not require any stains, contrast agents, or conductive coatings that can cause partial obstruction of the actual sample

Ability to image a variety of materials (i.e., nonconductive, magnetic, biological) under a variety of environmental conditions (i.e., ambient air, various gases, different humidity levels and temperatures)

AFM is a nondestructive method that allows the sample to be reused for additional analyses over time

Salient Features continued…

The precise nanometer control of the position and the picoNewton control of the force of the cantilever allows the physical manipulation of biomolecules, the dissection of biological structures the delivery of ligands, drugs or other materials to specific locations,and the precise measurement of interacting forces at specific sites;

The AFM apparatus can be modified by adding complementary methodologies such as the measurement of ionic conductance, total internal reflection fluorescence (TIRF), fluorescence resonance energy transfer (TIRF) and fluorescence imaging, microfluidics, physicochemical measurements that allow the measurement of structure and function of biological tissues among other modalities.

Abilities of AFM

Force measurment

Imaging

Manipulation

Basic components of Atomic Force Microscopy

• Piezoelectric scanner

• Flexible Cantilever with sharp probe

• Laser source

• Photodiode detector

• Feedback electronics

Principle Of AFM

• The AFM consists of a cantilever with a sharp tip (probe) at its end that is used to scan the specimen surface. When the tip is brought into proximity of a sample surface, forces between the tip and the sample lead to a deflection of the cantilever. This cantilever deflection is monitored by bouncing the laser beam on the back on the cantilever to a set of photodetectors which convert light signals into electrical signals.

APPLICATIONS

Drug-DNA complexes have

been studied with AFM to determine DNA ligand mode-

of binding.

In Nano Scale Drug Delivery System.

Imaging ion channels and

defining channelopathies

Imaging of DNA

• Watson & Crick model was based on the X-RAY Crystallography & Electron Microscopy

• In AFM there is no necessity to crystallize the DNA prior to imaging, and the possibility to study protein– DNA interactions and processes as they occur in normal cell

Drug DNA Complex

• This is of considerable interest since nucleic acid ligands are commonly used as anticancer drugs and in the treatment of genetic diseases

• AFM was used to study drug binding mode, affinity, and exclusion number by comparing the length of DNA fragments that have and have not been exposed to the drug.

• When exposed to ethidium, a well characterized inter-calator, the DNA strand was shown through AFM to have increased in length from 3300 nm to 5250 nm (see fig. 3).

Imaging ion channels and defining channelopathies

Amyloid ion channels and paradigm shift in amyloidogenic diseases• In a series of AFM studies, many amyloids, including amyloid beta protein,

amylin, alpha-synuclein,ADan, ABri, and Serum Amyloid A, form ion channel-like structures that show single ion channel activity when reconstituted in membranes has been depicted. Lin et al.2001;Quist et al.2005

• Channelopathies are a category of disease that traditionally have been thought of as being caused by altered function of channels or of the proteins that regulate channels, but amyloid deposition has actually created new channels that are likely pathologic.

• For instance, amyloid beta protein (1-42)induced changes in endothelial cells were tracked

Lin et al. 2001;Bhatia et al. 2000

• Furthermore, since the AFM operates under biological liquids,reagents can be added and the physiologic solutions changed during imaging. In this way, the therapeutic (preventive) capability of an agent can be evaluated directly and with nanometer resolution.

Amyloid beta channel induced cell death. Left half: Loss of synaptic arborization and neuronal death imaged with Calcein dye and fluorescence before and after addition of AβP to the buffer solution (compare panels A, C, E, and G (control)) with panels B, D, F, and G (after addition of nano-to-micromolar abeta peptide). Right half: Simultaneously,AFM can be used to image the cytoskeletal structure of cells to track effects of AβP on the cells, and to test various blocking agents for their performance in blocking toxicity effects. For details, see Lin et al. (2001) and Bhatia et al. (2000).

Nano Scale Drug Delivery System

• Nano medical Research is focused on design, Characterisation and delivery of Nano sized drug carriers:nanoparticles,liposomes,etc,.in the treatment of cancer.

• AFM-Directly observe very minute particles without any cumbersome and potentially contaminating sample preparation.

• By linking drug molecules to the tip, AFM can probe the target molecules on the cell surface.

• This technique is called single-molecule force spectroscopy (SMFS).

• By performing approach-retract cycles on the cell surface using drug-tethered tips, force curves can be recorded.

• The magnitude of the peak is equal to the binding force between a target molecule and a drug molecule.

The retract curve

Dynamic changes of cellular ultra-microstructure and elasticity in response to drugs

• Antimicrobial peptides are a promising class of antimicrobials that have demonstrated activity against antibiotic-resistant bacteria, parasites, viruses and fungi.

• AFM used to image the dynamic nanoscale changes on single living bacterial cells in response to CM15 (an antimicrobial peptide drug), revealing that the cell surface changed from smooth to corrugated after the stimulation of CM15. - Fantner et al(2014)

• AFM peak force tapping mode image revealed the effect of glyphosate and quercetin on single living keratinocyte cells showing that cells develop a filamentous cytoskeleton network after the stimulation of glyphosate, whereas the filamentous structures disappeared after the subsequent addition of quercetin.

-Heu et al(2012)

• Daunorubicin (Dau) is a drug used to treat several types of cancers, such as leukemia and neuroblastomas.

• Dynamic changes of DNA molecules in response to Dau as it intercalates in DNA to cause a local unwinding of the DNA was seen by using helix time-lapse AFM.

- Alonso-Sarduy et al(2013)

complement-mediated cytotoxicity (CMC) AFM images of lymphoma cells after the treatment of Rituximab

References

• Multidimensional atomic force microscopy for drug discovery: A versatile tool for defining targets, designing therapeutics and monitoring their efficacy by Ratnesh Lal , Morton F. Arnsdorf.

• Atomic force microscopy: A multifaceted tool to study membrane proteins and their interactions with ligands by Allison M. Whited, Paul S.-H. Park.

• AFM in Advanced Pharmaceutical Technology Nicolaos Scoutaris* and Dennis Douroumis* Pharmaceutica Analytica Acta.

• Mini Review on Feeling the forces: atomic force microscopy in cell biology by Jose´ Luis Alonso1, Wolfgang H. Goldmann*.

• Atomic force microscopy of macromolecular interactions by Christopher M Yip.

• Nanoparticle Delivery of Cancer Drugs by Andrew Z. Wang, Robert Langer, and Omid C. Farokhzad