Dr. Rajesh Karyakarte MD

Professor and Head,Department of Microbiology,

Government Medical College,

Akola, Maharashtra, India

Nanotechnology in Parasitology

Definition: NanotechnologyNanotechnology is the

understanding and control of matter at dimensions between approximately 1 and 100 nanometers, where unique phenomena enable novel applications. Encompassing nanoscale science, engineering, and technology, nanotechnology involves imaging, measuring, modeling, and manipulating matter at this length scale.

The National Nanotechnology Initiative, US.

Atomically precise positioning of carbon monoxide molecules on a copper surface enables data storage with bits smaller than atoms

Nanotechnology: Timeline

The physicist Richard Feynman first developed the concept 'nanotechnology' (but he did not specifically use this term) in a talk “There's Plenty of Room at the Bottom,” given at an American Physical Society meeting at Caltech on December 29, 1959. The Nobel Prize in Physics 1965 was awarded jointly to Sin-Itiro Tomonaga, Julian Schwinger and Richard P. Feynman "for their fundamental work in quantum electrodynamics, with deep-ploughing consequences for the physics of elementary particles".

Nanotechnology: Timeline cont.…

Professor Norio Taniguchi of the Tokyo Science University, introduced the term “nanotechnology”, in a 1974 paper. He described nanotechnology as the processing of, separation, consolidation, and deformation of materials by one atom or by one molecule." N. Taniguchi, "On the Basic Concept of 'Nano-Technology'," Proc. Intl. Conf. Prod. Eng.

Tokyo, Part II, Japan Society of Precision Engineering, 1974

Nanotechnology: Timeline cont.…

In the 1980s, Dr. K. Eric Drexler, promoted nanoscale phenomena through books:• Engines of Creation: The Coming Era of

Nanotechnology• Nanosystems: Molecular Machinery,

Manufacturing, and Computation He was ultimately responsible for the term nanotechnology to acquire its current sense.

Three Champions of Nanotechnology

Feynman Taniguchi Drexler

Nanotechnology developed in early 1980s with two major developments; the birth of cluster science and

the invention of the scanning tunneling microscope (STM).

Nanotechnology: Early development

In physics, the term clusters denotes small, multi-atom particles.

Cluster science: studies the gradual development of collective phenomena which characterize a bulk solid.

Collective phenomena (color, electrical conductivity, and magnetic properties) break down for very small cluster sizes.

Cluster science

Scanning tunneling microscope (STM): is an instrument for imaging surfaces at the atomic level.

Its development in 1981 earned its inventors, Gerd Binnig and Heinrich Rohrer (at IBM Zürich), the Nobel Prize in Physics in 1986

Scanning tunneling microscope

The field of nanotechnology matured with the discovery of fullerenes in 1985 and carbon nanotubes a few years later.

Nanotechnology: Further developments

Carbon NanotubeBuckminsterfullerene C60

The Atomic Force Microscope was invented in 1986. It allowed for unprecedented control over nanomaterial design and characterization

Nanotechnology: Timeline cont.…

A nanometer is one-billionth of a meter. A single gold atom is about 1/3 of a

nanometer in diameter. A DNA double helix has a diameter of about

2 nm.Picornavirus is around 20 nm. Mycoplasma is around 200 nm in.A sheet of paper is about 100,000 nm thick. Fascinatingly, the beard of a man grows by

a nanometer in the time he takes to bring the razor to his face for a shave

Scale of things - Nanometer

Nanotechnology in ParasitologyAtomic Force

Microscope (AFM) has a fine pointed tip attached to a cantilever that moves over or touches the sample.

The cantilever deflects as the tip is pulled toward or pushed away from the surface.

A laser is bounced off the mirrored backside of the cantilever onto a photodiode to measure this deflectionFrom Merz et al., Nature 407:98, 2000.

Optical Tweezers

Optical Tweezers Continued… (1)Optical tweezers allow

manipulation and simultaneous observation of biological processes of living microorganisms, as flagellate protozoan.

Optical tweezers have been used to apply forces in the pN (piconewtons) and to measure displacements in nanometers (nm) of a range of objects ranging in size from 10 nm to over 100 mm.

Optical Tweezers Continued… (2)

Optical Tweezers have been used:To insert DNA in cells and fertilization in vitro;To measure and compare cell displacements; To measure forces of cardiac muscle fibers; To measure the length of a DNA molecule; To study motility of human spermatozoa; To detect antigens at femtomolar level;To study bacterial flagella motorsTo study strength, elasticity and viscosity of

RBCs;To study chemotaxis of Leishmania

amazonensis and Trypanosoma cruzi.

Optical Tweezers Continued… (3)Thomaz et al (2009) measured the propulsion forces of

the flagellum of T. cruzi .When T. cruzi is more than 50 μm away from the

midgut cells of reduviid bug (Rhodnius prolixus). It showed an erratic movement.

When less than 20 μm away from the midgut cells, T. cruzi moved towards the cells.

Maximum propulsive force of the flagellum was 0.8 pN.

Nanoscopy

Nanoscope

Nanoscope (AFM) can help in studying living cells in gaseous and liquid environments.

Nanoscopes that are dedicated to biological applications are available commercially.

Nanoscopes have many scan modes for analysis. The intermittent contact

mode produces topological information.

Phase imaging allows for the analysis of adhesive and elastic properties, in addition.

In parasitology, AFM was used to study the structural organization of trypanosomatid parasites by Dvorak and collaborators almost 10 years ago.

This study was successful in drawing attention of the parasitologists towards the potential of AFM.

The published images however did not add significant new information regarding the structure of these parasites.

Nanoscopy Continued… (1)

Dvorak, J. A., Kobayashi, S., Abe, K., Fujiwara, T., Takeuchi, T. and Nagao, E. (2000) The application of the atomic force microscope to studies of medically important protozoan parasites. J. Elec. Microsc. 49, 429–435.

Rocha et al, 2008 using similar methodology but with an additional pretreatment step of treating the protozoal cells with detergent, produced images wherein both the cell surface and the intracellular structures of Trypanosoma cruzi could be well recognized.

Nanoscopy Continued… (2)

Rocha, G. M., Miranda, K., Weissmüller, G., Bisch, P. M., de Souza, W. (2008) Ultrastructure of Trypanosoma cruzi revisited by atomic force microscopy. Microsc. Res. Tech. 71, 133–139.

AFM image: flagellum of slightly detergent-extracted T. cruzi. A furrow along the major axis of the flagellum with periodically organized protrusions can be seen (arrowheads).

Joergensen et al (2010) studied the kinetics of antibody binding to VAR2 Chondroitin Sulfate A Plasmodium falciparum erythrocyte membrane protein 1 antigen.

Molecular modeling after nanoscopy indicate that: PfEMP1 has a large size The architecture of the knobs

facilitates cytoadhesionBut reduces avidity of antibody-PfEMP1 binding.

Nanoscopy Continued… (3)

Joergensen LM, Salanti1 A, Dobrilovic T, Barfod L, Hassenkam T, Theander TG, Hviid L, Arnot DE. The kinetics of antibody binding to Plasmodium falciparum VAR2CSA PfEMP1 antigen and modelling of PfEMP1 antigen packing on the membrane knobs. Malaria Journal 2010, 9:100.

Nanoscopy Continued… (4)

AFM derived measurement of the dimensions of the knob structures on the infected erythrocyte membrane.

Two Knobs

Two-color enhanced three dimensional imaging

Topology measurements of the surface bisected by the red line shown in Figure B.

The knob in the infected RBC shown previously is 120 nm in diameter and 24 nm high and its surface area is around 13,000 nm2.

An estimated maximum of 110 VAR2CSA molecules could be tightly packed onto a knob of the dimensions shown in the figure.

Nanoscopy Continued… (5)

Knobs form at actin-spectrin-ankyrin junctions and inter-knob distances are related to the regular inter-junction distances (100-200 nm).

This makes antibody crosslinking of PfEMP1 between different knobs on the same erythrocyte by IgG1 (or even IgM) antibodies effectively impossible.

Nanoscopy Continued… (5)

Nanoscopy Continued… (5)Actin-spectrin-ankyrin junctions

• The erythrocyte membrane skeleton is organized as a polygonal network formed by five to seven extended spectrin molecules linked to short actin filaments approximately 40 nm in length .

• The spectrin-actin network of erythrocytes is coupled to the membrane bilayer primarily by association of spectrin with ankyrin, which in turn is bound to the cytoplasmic domain of the anion exchanger

Quantum dots (QDs)

• The light emitting Quantum dots consist of semiconductor nano-crystals that are 1 to 10 nm in diameter.

• QDs resist photobleaching and have higher absorption coefficients than fluorophores.

• QD particle size determines wavelength of the emitted light.

• By changing the sizes of QDs it is possible to distinguish among different classes of target molecules simultaneously, while using a single excitation wavelength.

Quantum dots (QDs)

Quantum dots (QDs) Continued… (1) • Feder et al (2009) used Green –emitting

cadmium telluride quantum dots (CdTe QDs) and Yellow-emitting cadmium selenide quantum dot (CdSe QDs) to label T. cruzi.

• These QDs (0.2 μM) had no effects on the development of T. cruzi.

• Further, due to the high photostability of the QDs, in vivo imaging of long-term interaction between T. cruzi and live cells of Rhodnius prolixus was possible Feder D, Gomes SAO, de Thomaz A.A, Almeida DB, Faustino WM, Fontes A.,

Stahl CV, Santos-Mallet JR, Cesar CL. In vitro and In vivo Documentation of Quantum Dots Labeled Trypanosoma cruzi & Rhodnius prolixus Interaction using Confocal Microscopy. Parasitology Research. 2009;106 (1):85-93.

Quantum dots (QDs) Continued… (2)

In vitro Interaction Assay showing Trypanosoma cruzi adhering to the midgut epithelium of Rhodnius prolixus by fluorescent labeling with green emitting CdTe quantum dots to acquire 3 frames per second confocal fluorescence images (After Feder et al., 2009).

Nanotechnology and Drug Delivery

Drug Delivery Continued… (1)

Nanotechnology in drug delivery and targeting. The major components are either lipid or polymers (After Couvreur and Vauthier, 2006)

Drug Delivery Continued… (2)

• Liposomes were proposed in 1974 by Gregoriadis et al for drug delivery. • Use of nanodevices, particularly

liposomes, has reduced the toxicity of amphotericin B 50- to 70-fold in leishmaniasis. • Thus, more drug ( 5-fold) can be

administered.• The liposome formulation, was

marketed in 1996 under the brand name AmBisome®.

In addition to reduction of the toxicity, these nanosized liposomes are not immediately cleared by the macrophages present in liver and spleen.

Thus, majority of these liposomes carrying amphotericin B remain in the blood circulation and also achieve a high enough concentration in infected tissues.

This nanodevice formulation has one more advantage of killing both phagocytized and non-phagocytized parasites.

Drug Delivery Continued… (2)

Drug Delivery Continued… (3)The main problem with AmBisome® is its high cost.To overcome this problem, nanodisks (250 nm in

diameter, 3 nm in thickness) that require far less lipids have been developed.

Thank You!