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S C H O O L O F P U B L I C H E A L T H & Z O O N O S E S

Nanoparticles and Animal Health

NanotechnolNanometre

Nano-

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Nanomedicine

NanoscienceNanobotsA prefix that means very,

very, small.

The word nano is from the Greek

word ‘Nanos’ meaning Dwarf. It is a

prefix used to describe "one billionth"

of something, or 0.000000001.

Nano:

How Small Is Nanoscale?

What is Nanotechnology?

A disruptive technology, with a potential to changethe world as we know it today.

Nanotechnology? It is the study of controllingand manipulating matter on an atomicand/or molecular scale.

It deals with structures the size of 100nanometers or smaller in at least one dimension.

It’s a very diverse technology

Definitions

A nanometer is a billionth of a meter.

It’s difficult to imagine anything so small, think of something only 1/80,000the width of a human hair.

Ten hydrogen atoms could be laid side-by side in a single nanometer.

Nanotechnology is the creation of useful materials, devices, and systemsthrough the manipulation of matter on this miniscule scale.

The emerging field of nanotechnology involves scientists from manydifferent disciplines, including physicists, chemists, engineers, andbiologists.

There are many interesting nano devices being developed that have apotential to improve cancer detection, diagnosis, and treatment

National Nanotechnology Initiative

The National Nanotechnology Initiative, a government initiative inthe USA describes nanotechnology as: ‘research and development(R&D) aimed at understanding and working with – seeing,measuring and manipulating – matter at the atomic, molecular andsupramolecular levels. This correlates to length scales of roughly 1 to100 nanometres. At this scale, the physical, chemical and biologicalproperties ofmaterials differ fundamentally and often unexpectedlyfrom those of the corresponding bulk materials’.

Nanotechnology, as a new enabling technology, has the potential torevolutionise agriculture and food systems throughout the world.Nanotechnology can provide new tools for molecular and cellularbiology and new materials for pathogen detection, so there areseveral areas in which nanotechnology could be applied to thescience and engineering of agriculture and food systems, e.g.agricultural and food systems security, disease treatment deliverysystems, and the protection of the environment

History of Nanotechnology• ~ 2000 Years Ago – Sulfide nanocrystals used by Greeks and Romans to dye

hair

• ~ 1000 Years Ago (Middle Ages) – Gold nanoparticles of different sizes used

to produce different colors in stained glass windows

• 1974 – “Nanotechnology” - Taniguchi uses the term nanotechnology for the

first time

• 1981 – IBM develops Scanning Tunneling Microscope

• 1985 – “Buckyball” - Scientists at Rice University and University of Sussex

discover C60

• 1986 – “Engines of Creation” - First book on nanotechnology by K. Eric

Drexler. Atomic Force Microscope invented by Binnig, Quate and Gerbe

• 1989 – IBM logo made with individual atoms

• 1991 – Carbon nanotube discovered by S. Iijima

• 1999 – “Nanomedicine” – 1st nanomedicine book by R. Freitas

• 2000 – “National Nanotechnology Initiative” launched

A Brief History of Nanotechnology

On December 29, 1959, physicist Richard Feynman gave a radical lecture at an American Physical Society meeting at Caltech titled “There’s Plenty of Room at the Bottom”.

Feynman suggested that it should be possible to make machines at a nano-scale that "arrange the atoms the way we want", and do chemical synthesis by mechanical manipulation.

This lecture was the birth of the idea and study of nanotechnology.

Hibbs’s Idea on Nanotechnology in Medicine

Albert R. Hibbs -a noted mathematician was fascinated by self-actuated machines. According to Feynman, Hibbs originally suggested to him (circa 1959) the idea of a medical use for Feynman's theoretical micromachines:“A friend of mine (Albert R. Hibbs) suggests a very

interesting possibility for relatively small machines. He says that… it would be interesting in surgery if you could swallow the surgeon. You put the mechanical surgeon inside the blood vessel and it goes into the heart and ``looks'' around … It finds out which valve is the faulty one and takes a little knife and slices it out. Other small machines might be permanently incorporated in the body to assist some inadequately-functioning organ”. – Richard Feynman, “There’s Plenty of Room at the Bottom”.

What Feynman and Hibbs considered a possibility, today 51 years later, is becoming a reality.

Various Types of Nanomaterials

10

Anthropogenic or Engineered NPs

Incidental Particles from:

Natural Particles from:

•Carbon-based•Nanotubes,•Fullerenes•Metal Oxides•Quantum Dots•Nanotubes•Nanowires•Dendrimers

•Combustion products•Industrial Processes•Vehicles emissions•Construction

•Plants, Trees•Oceans, other•water bodies•Erosion•Dust

Types of Nanoparticles• Nanoparticles fall into three major types:

• Naturally occurring• Incidental• Engineered

• Naturally Occurring• Examples of naturally occurring nanoparticles include:

• Sea spray• Mineral composites• Volcanic ash• Viruses

• Incidental Nanoparticles• A result of man-made industrial processes, incidental nanoparticles include:

• Cooking smoke• Diesel exhaust• Welding fumes• Industrial effluents• Sandblasting

• Engineered Nanoparticles• Engineered nanoparticles comprise of any manufactured particles with nanoscale

dimensions. Examples include:• Metals• Quantum dots• Buckyballs/nanotubes• Sunscreen pigments• Nanocapsules

The size of selected nanotechnology materials is

estimated to be as follows:

Nanoparticles 1 – 100 nm

Fullerene (C60) 1 nm

Quantum Dot 8 nm

Dendrimer 10 nm

Materials found in nature are typically referenced to

have the following dimensions:

Atom 0.1 nm

DNA (width) 2 nm

Protein 5 – 50nm

Virus 5 – 100nm

Bacteria 1,000 – 10,000 nm

White Blood Cell 10,000 nm

Creation of Nanoparticles

Two basic approaches for creating nanodevices.

Top-down approach Bottom-up approach

Top-Down

Milling processes: Themechanical production approachuses milling to crushmicroparticles.

This approach is applied inproducing metallic and ceramicnanomaterials.

For metallic nanoparticles, high-energy ball mills are used. Suchmills are equipped with grindingmedia composed of wolframcarbide or steel

Bottom-up

Vapour condensation: typicallyused to make metallic oxide ceramicnanoparticles.

A solid metal is evaporated and isthen rapidly condensed to formnanosized cluster that settle down inform of powder.

Or, the metal vapour is released intoa vacuum chamber which contains arotating drum coated with a thinlayer of viscose substance.

Nanoparticles form in suspension inthe liquid coating on the drum.

Nanodevices Are Small Enough to Enter Cells

Most animal cells are 10,000 to 20,000 nanometers in diameterNanoscale devices (less than 100 nm) can enter cells and the

organelles inside them to interact with DNA and proteins. Tools developed through nanotechnology may be able to detect

disease in a very small amount of cells or tissue.They may also be able to enter and monitor cells within a living

body.

Nanomaterials categories

One dimension

Less than 100nm

Nanoscale layersEg. thin films or surface coatings like computer chips

Two dimensions

Nanowires and nanotubes

Three dimensions

Precipitates

Colloids and Quantum dots (tiny particles of semiconductor materials)

Nanoparticles preparation

Nanoparticles prepared from such substances asproteins, olysaccharides and synthetic polymers

The selection of matrix materials is dependent on

a) Size of nanoparticles required

b) Inherent properties of the drug, e.g., aqueoussolubility and stability

c) Surface characteristics such as charge andpermeability

d) Degree of biodegradability, biocompatibility andtoxicity

e) Drug release profile desired

f) Antigenicity of the final product

Types of nanoparticles

Nanomaterials are materials (either newly created throughnanotechnology or that exist in nature) that provide the potential to manipulate structures or other particles at the nanoscale and to control and catalyse chemical reactions.

Materials are generally composed of particles of many sizes. The shape, structure and aggregation of particles at the nanoscale influence the properties of the material at the macro-level.

Specific examples of nanomaterials are buckeyballs, dendrimers, nanoshells, nanotubes and quantum dots.

Liposomes, polymer nanoparticles (nanospheres and nanocapsules)

Solid lipid nanoparticles, nanocrystals, polymer therapeutics such asdendrimers, fullerenes (most common as C60 or buckyball, similar in size ofhormones and peptide a-helices)

Inorganic nanoparticles (e.g. gold and magnetic nanoparticles)

Bucky balls (fullerenes) Fullerenes, a carbon allotrope The buckminster fullerene is the most common form of fullerene 7 Å in diameter with 60 carbon atoms arranged in a shape known as

truncated icosahedrons It resembles a soccer ball with 20 hexagons and 12 pentagons

Scientists (6) have discovered how to make the metal-filled buckeyballssoluble, bringing them a step closer to biological applications, such as the delivery of medicine or radioactive material to a disease site

The idea of using the 60-atom to 80-atom hollowcarbon molecules for drug delivery is what gives addedbiological functionality to a buckeyball. The aim is toattach water-soluble groups of peptides or hydrophilicchains to get these molecules into the blood stream.

Nanotubes…

Nanotubes - opened on two sides with additional atom groups added in the characteristic hexagon shape to form a hollow carbon tube (cylinder)

Sheet of graphite (a hexagonal lattice of carbon) rolled into a cylinder

This nanotubes are used to tracking oestrus in animals - detect the estradiol antibody at the time of oestrus by near infrared fluorescence

Used in gene therapy

Another nanodevice that will help identify DNA changes associated with cancer is the nanotube.

Nanotubes are carbon rods about half the diameter of a molecule of DNA that not only can detect the presence of altered genes, but they may help researchers pinpoint the exact location of those changes.

To prepare DNA for nanotube analysis, scientists must attach a bulky molecule to regions of the DNA that are associated with cancer. They can design tags that seek out speciÞcmutations in the DNA and bind to them.

nanotubes

Dendrimers…

Dendrimers are 3-D man-made nanomolecules with regular branching structures

The branches arise from the core in shape of a spherical structure by means of polymerisation

This results in formation of cavities within the dendrimermolecule which can be used for drug transport

The ends of the dendrimer molecule can be attached with other molecules for transport

Quantom dots…

A 2-10 nm nano-scale crystalline structure made from cadmium selenide

Re-emits the white light in a couple of nanoseconds -specific color which can be made to fluoresce when stimulated by light

Their structure consists of an inorganic core, the size of which determines the colour emitted, an inorganic shell and an aqueous organic coating to which biomolecules are conjugated

These particles enable powerful new approaches to genetic analysis, drug discovery, and disease diagnostics

…Quantom dots…

…Quantom dots

Quantum dots - emit light at any wavelength

Inserted almost anywhere, including liquid solution, dyes etc

Quantum dots can be attached to a variety of surface ligands, and inserted into a variety of organisms for in-vivo research

quantum dots respond to light- it may be possible to illuminate the body with light and stimulate the quantum dot to heat up sufficient to kill the cancerous cell

Nano shells

Dielectric (silica) core coated with an ultra-thin metallic(gold), layer size ranging from 10-500 nm.

Strong optical absorption.

Optical response depends on size of the core and thickness of the gold shell.

Shows broad range of an optical spectrum.

Nanoshells are extremely small beads of glass coatedwith gold. They can be fashioned to absorb light ofalmost any wavelength, but nanoshells that captureenergy in the near-infrared, which can easily penetrateseveral centimeters of tissue

Biosilicon

Highly porous silicon based nanomaterial product, that canrelease a medicine slowly over a period of time.

First by Australian company Sivida ,they fashion tinycapsules(to be swallowed) and also tiny needles that can bebuilt into patch to invisibly pierce the skin and deliver drugs.

Monitor sugar level in the blood.

Viral nanoparticles

Viruses including cowpea mosaic virus, cowpea chloroticmottle virus, canine parvovirus, and bacteriophages have beenused in tissue targeting and drug delivery.

ligands or antibodies including transferrin, folic acid, andsingle-chain antibodies have been conjugated to viruses for

specific tumor targeting in vivo.

( Manchester M et al 2006)

canine parvovirus, have natural affinity

transferrin receptors that are up-regulated on a variety

of tumor cells. (Singh P et al. 2006)

Applications

Applications in Surgery

With nanotechnology, minute surgical instruments and robots can be made which can be used to perform microsurgeries on any part of the body.

Instead of damaging a large amount of the body, these instruments would be precise and accurate, targeting only the area where surgery should be done.

Visualization of surgery can also be improved. Instead of a surgeon holding the instrument, computers can be used to control the nano-sized surgical instruments. “Nanocameras” can provide close up visualization of the surgery

Less chance of any mistakes or faults Surgery could also be done on tissue,

genetic and cellular levels.

Applications in Medical Robotics

Nano-robotics, although having many applications in other areas, have the most useful and variety of uses in medical fields.

Potential applications include early diagnosis and targeted drug delivery for cancer, biomedical instrumentation, surgery, pharmacokinetics, monitoringof diabetes, and health care.

Future medical nanotechnology expected to employ nanorobots injected into the patient to perform treatment on a cellular level.

Drug delivery

By only targeting the afflicted cells, less of the drug isneeded, reducing the side effects and making the drugless expensive.

As drug only needs to go to certain targets instead ofwhole body, it works faster to relieve the patient.

Smaller the drug–carrying unit, more it tends toconcentrate itself in inflamed areas.

By using the nanobiotechnology ,drug delivery can beaccomplished by encapsulation of the drug inside amembrane with channels that open and closeaccording to outside stimuli.

Applications in Drugs and Medicine

Nanotechnology can deliver medicine or drugs into specific parts of the human body, thereby making them more effective and less harmful to the other parts of the body.

A recent study conducted by NIH found anti-cancer gold nanoparticles very effective.

Gold “nanoshells” are useful to fight cancer because of their ability to absorb radiation at certain wavelengths. Once the nanoshells enter tumor cells and radiation treatment is applied, they absorb the energy and heat up enough to kill the cancer cells.

Not only gold but other elements can also be used.

Disease diagnosis and treatment

Diagnosis and Imaging: Microchips labelled with humanmolecules are designed to emit an electrical impulse signal whenthe molecules detect signs of a disease.

Special sensor nanobots cheap and portable.

Inserted into the blood, check and warn of any possible desease.

Quantum dots: bind themselves to proteins unique to cancercells, kill the cancer cells by exposing UV light.

Nanoshells:injected into theanimal’s loodstreamwith targeted agents.

Attach to the surfacereceptors of cancercells. Illumination ofthe body withinfrared light raisesthe cell temperatureto about 55°C, which‘burns’ and kills thetumour.

Cancer-battling nanoshellsThe nanoshell has a gold exterior layer which covers interior

layers of silica and Drugs. It can release tumor- specific antibodies when infrared light is administered

‘Smart’ superparamagneticnanoparticles:

• when injected in the bloodstream target tumour receptor cells.

made from iron oxides (5-100 nm), whensubjected to a magnetic field enhance theability of the nanoparticles to locatetumour cells.

At the site of the tumour the nanoparticlesemit an attached drug to kill the cancercells.

Gold nanoparticles: Shows intense color invisible region for spectroscopic detection.

Used in biological labeling and imaging.

Can be prepared easily; low toxicity.

Gold reflects red light at nanoscale,

thus it is used to kill the cancerous

cell with visible light without

harming the normal cells.

(Zahrov V P et al 2005)

Nanochips: employs the power of an electroniccurrent that separates DNA probes to specificsites on the array based on charge and size.

the test sample (blood) can be analyzed for targetDNA sequences by hybridization with theseprobes.

Hybridised DNA will fluoresce which isdetected an relayed back to an onboard systemthrough platinum wiring.

MEMS Methods of making micro-sized machines or microelectromechanical systems(MEMS) are already established. Fully functional pumps, rotors, sensors and

levers exist at the microscale. swallowed capsule technology pills that allow

doctors to visualize GI bleeding.

“The patient swallows a capsule containing alightemitting diode for illumination, a CMOS(complementary metal-oxide semiconductor)video camera and optics for taking images, abattery, and a transmitter”

The images are then transmitted to a receiverworn on the patient’s belt and the doctor isthen able to diagnose the cause of theailment.

Tissue reconstruction

Treatment of an Injured Bone:

An ultrasound is performed on existing bonestructures and then bone-like nanoparticles arecreated using the results of the ultrasound.(Silva et al 2004).

The bone-like nanoparticles (15-18 nm ceramic andpoly methyl methacrylate copolymer) are inserted intothe body in a paste form. (Adhikari et al2005).

When they arrive at the fractured bone, they assemblethemselves to form an ordered structure which laterbecomes part of the bone. (Adhikari et al 2005).

Treatment of an injured nerves:

Samuel Stupp and John Kessler at NorthwesternUniversity in Chicago have made tiny rod like nano-fibers called Amphiphiles.

They are capped with amino acids and are known tospur the growth of neurons and prevent scar tissueformation.

(Wiess et al 2005)

Artificial RBC’s: ultrathin polyethyleneglycolpolylacetic acid (PEG-PLA) membranecontaining Hb and enzymes.

(Chang et al 2009)

Applications to animal health

Nanotechnology has opened up new vistas for applications in molecularbiology, biotechnology and almost all the disciplines of veterinary and animalsciences.

Excellence in animal health and production can be achieved by translation ofthis newer technology to create effective services and products for animals.

The ability to manufacture and manipulate matter on the nanoscale hasoffered opportunities for application in diverse areas of animal sciences.

Nanosensors, nanovaccines, adjuvants, gene delivery and smart drug deliverymethods have the potential to revolutionize animal health and production.

There can be numerous applications of the nanomaterials for disease diagnosis,treatment, drug delivery, animal nutrition, animal breeding, reproduction,tissue engineering and value addition to animal products

Applications to animal health…

“Smart” drug delivery system Disease diagnosis and treatment Gene therapy or DNA delivery Drugs discovery Nanovaccines and vaccine adjuvants Tissue repair Identity preservation and quality assurance Animal breeding and reproduction Animal nutrition Value added to animal products

Smart drug delivery system…

The development of ‘smart’ treatment delivery systems onthe nanoscale uses similar concepts applied at themolecular level.

For example, ‘smart’ drug delivery systems in animalswould most likely contain small, sealed packages of thedrug to be delivered.

The packages would not be opened until they reach thedesired location in the animal, e.g. the site of infection.

…Smart drug delivery system

Advantages of Drug delivery system

Time-controlled

Spatially Targeted

Self-regulated

Remotely Regulated

Pre-programmed

Antimicrobial property

The silver nanoparticles show efficient antimicrobial property compared to other salts

Most effective on E.Coli, S.aureus, Klebsiella, Pseudomonas

The nanoparticles preferably attack the respiratory chain, cell division finally leading to cell death

The STEM (Scanning Transmission Electron Microscopy) confirms the presence of silver in the cell membrane and inside the bacteria

Silver nanoparticles in most studies are suggested to be non-toxic. But it suggested to be hazardous to the environment (Braydich-Stolle et al., 2005)

Early detection of cancer…

The current systems are limited by their selectivity and efficiency to concentrate rare cells for molecular assays

Nanoscience can detect - circulating cancer cells, which present often at 1–2 cells per milliliter of blood.

Combinatorial use of magnetic nanoparticles and semiconductor QDs - increase the ability to capture and evaluate these rare circulating cancer cells

Bionanobarcodes, nanocantilevers, and nanowiresare promising technologies

Nanobarcode

Cancer cells detection

Protein and nucleic acid detection based on biobarcode-amplification

Gold nanoparticles are modified with both target capture strands and bar code strands that are subsequently hybridized to bar code DNA, and magnetic microparticlesmodified with target capture strands (BCA)

Gold nanoparticles and the magnetic microbeads form sandwich structures that are magnetically separated from solution.

Unhybridized bar code DNA are removed

The bar codes (hundreds to thousands per target) are detected by using a colorimetric method

Quantom dots on cancer detection…

QD staining provides spatial localization information (both inter- and intracellular),

QD probes are delivered to tumors by both a passive targeting mechanism and an active targeting mechanism

In the passive mode, macromolecules and nanometer-sized particles are accumulated preferentially at tumor sites through the Enhanced permeability and retention (EPR)effect.

For active tumor targeting, Gao et al. used antibody conjugated QDs to target a specific membrane antigen.

Detection of tumor

Cancer therapy

Imaging & therapy for tumors

Multifunctional nanoparticles for integrated cancer imaging and therapy

Quantom dots on cancer detection…

Nanovaccines…

Vaccines require immunostimulating compounds, adjuvants, which act nonspecifically to increase the immune response to a defined antigen

Nanometer adjuvants are

1. Liposome

2. ISCOM based adjuvant

3. Biobullets

4. Virus like particles

Nano-particles - 40–50 nm - potential to induce potent cell mediated (CD4 and CD8 T cells) as well as humoral immune responses

…Nanovaccines

VLP vaccine against BT & AHS – strong protection

ISCOM based vaccines effective on H5N1 in chickens and EHV - 2 in horses

Liposomes added vaccines protect the cattle against BVDV

Liposomes have also been used to deliver allergen extracts as immunotherapy for refractory canine atopic dermatitis

“Biobullets” made of photopolymerized PEG hydrogels can serve as biodegradable bullets used to wild animals for vaccination. Eg. Bruellaabortus

Some of the proteins, polysaccharides synthetic polymers and lipids used as

nanocarrier for drug delivery.

Nanoformulations against infectious organisms tested for drug delivery in experimental animals with

potential for veterinary use. (Manuja eta l., 2012)

Nanovaccines/adjuvants against infectious organisms tested in

experimental animals with potential for veterinary use.

(Manuja et al., 2012)

Examples

Disease diagnosis: A rapid, sensitive test has been developed for detection ofFMD virus which relies on the sensitivity and movement of liquid crystals at thenanoscale in the presence of a target molecule. Virus binding in a detectionregion is identified by changes in liquid crystal orientation.

Therapeutics: the common antibiotic molecule gentamicin was bound to ahydrogel using a peptide linker which can only be cleaved by a protease enzymeformed by Pseudomonas aeruginosa; thus, the antibiotic is not released inabsence of the organism Proteases specific to particular bacteria can be used asthe signal for the release of different spectra of antibiotics from the samematrix depending on the strain of bacterium.

Animal nutrition: A nanocomposite of MgO-SiO2 has been used as an effectiveadsorbing agent for removal of aflatoxin from wheat flour. Similarly, a modifiedmontmorillonite nanocomposite (MMN) has been used to reduce the toxicitydue to aflatoxin in feeds of broiler chicks.

Value addition to animal products: Nanoparticles are being used to removeCampylobacter and E. coli from poultry products. Listeria monocytogenes,another foodborne pathogen was detected in spiked milk samples by magneticnanoparticle-based immune-magnetic separation combined with real-timePCR.

Safety and toxicological issues Aggregates of nanoparticles are water soluble and kill useful

bacteria. (Balbus et al 2005).

Nanoparticles are very light and can easily become airborneand can cause asthma, bronchitis and can be fatal.(Donaldson et al 2004).

Nanoparticles flowing thorough the bloodstream may affect theclotting system. (Donaldson et al 2004).

May damage the brain and nervous system, could be fatal.

Might move through a mother’s placenta to the foetus.

(Howard V 2004)

Nanoparticles used in sunscreens created free radicals thatdamage DNA.

Conclusions

Nanotechnology is still in its early stages.

As further research continues in this field, more treatments willbe discovered.

Many diseases that do not have cures today may be cured bynanotechnology in the future.

If everything runs smoothly, nanotechnology will one day become part of our everyday life and will help save many lives.

“Any intelligent fool can make things bigger, more complex and more violent. It takes a genius- and a lot of courage- to move in the opposite direction.”-

Albert Einstein

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