Nanotechnology and Infectious Diseases

NANOTECHNOLOGY IN
INFECTIOUS DISEASES
Major Inam Danish Khan
Dept of Microbiology and Molecular Medicine
Army Hospital Research and Referral, New Delhi

PROLOGUE
 Golden ages of Microbiology
 Introduction
 History of nanotechnology
 Scope of nanotechnology
 Visualization
 Diagnostics
 Nano-engineered products
 Emerging nano-concepts
 Nanotechnology issues

GOLDEN AGES OF MICROBIOLOGY
 First Golden Age
 Bacterial physiology and nutrition
 Cultivation methods
 Immunology, phagocytosis
 Antibodies
 Vaccines
 Viruses
 Second Golden Age
 Cellular immunology
 Monoclonal antibodies
 Transposons
 Genetic engineering
 Nucleic acid sequencing
 Protein technology
 Third Golden Age
 Emerging infectious diseases
 Identification of uncultivated microbes
 Role of microbes in modulating host development
 Role of microbes in chronic diseases
 Rapid identification of microbes
 Use of microbes as NANOMACHINES

INTRODUCTION
 Nanoscience encompasses the common unifying concepts and
physical laws that prevail in the nanoscale
 1 nanometer = 10-9 meter (1 billionth of a meter)
 Greek ‘nanos’ or Latin ‘nanus’ means dwarf
 Physical, chemical, optical and electrical properties differ
 Nanotechnology is the understanding and control of matter at
dimensions in the nanoscale
 Nanotechnology consists of the process of separation, consolidation
and deformation of materials by one atom or one molecule
 Nanotechnology is the creation of functional materials, devices and
systems, through the understanding and control of matter at
dimensions in the nanometer scale length (1-100 nm), where new
functionalities and properties of matter are observed and harnessed
for physical, chemical and biological interest

NANOTECHNOLOGY TERMS
 Cluster - A collection of up to 50 units (atoms or molecules)
 Colloid particle - 1-1000 nm particle in liquid phase
 Nanoparticle - 1-100 nm particle that could be noncrystalline,
aggregate of crystallites or a single crystallite
 1st generation Nanoparticles: <100nm, 2nd generation Nanoparticles: <10nm
 Nanocrystal - Single crystal in the nanometer range

HISTORY OF NANOTECHNOLOGY
 ~2000 BC: Sulfide nanocrystals used by Greeks & Romans to dye hair
 Photography and catalysts
 1959: R. Feynman - There's Plenty of Room at the Bottom
 1974: Taniguchi
 1981: Scanning Tunneling microscope
 1985: Buckyballs (Buckminster fullerenes) – R Kroto
 1986: Atomic Force Microscope
 1991: Carbon nanotube - S.Lijima
NANOTECHNOLOGY IN INFECTIOUS DISEASES
 Diagnostics
 Monitoring
 Therapeutics
 Surveillance
 Prevention
 Research

SCOPE OF NANOTECHNOLOGY
 Nanotechnology is any technology which operates in the nanoscale
 Exploring the methodology of nano-operations
 Physics, chemistry and biology of nanotechnology
 Enabling visualization and studies in the nanoscale
 Biomolecular interactions, pathogen interactions
 Equipments - EM, FIBM, DBM, SPM – STM & AFM
 Designing and construction of nano-engineered products
 Biological nanoparticles, synthetic nanomaterials, biosensors
 Detection and monitoring of deployed technology
 Disease surveillance
 Equipments - TIRM , Optical nanoscopy, SERS
 Nanotechnology applications in disease diagnosis
 Nanoarray, quantum dots, Lab on a chip
 Nanotechnology in targeted therapies/preventive interventions
 Pharmacy on a chip, targeted drug delivery, DNA vaccines
 Research on nanoparticles

ELECTRON MICROSCOPY
 Use of electron beam (shorter wavelength than light) to produce images
 Source of illumination – Heated tungsten filament, cathode, electron gun
 Electron cloud propelled by high volatge 50-100 kV
 High vacuum of the order of 10-6 mm Hg is maintained
 Magnification 10000X to 100000X
 Copper grid 2-3 mm dia - Mesh size 200 for tissue & 400 for microbes
 EM lenses
 Limitations
 Sample has to be dry/dead
 Electrons are ionizing and
may damage the specimen
 Accumulated electrons in the
sample repel electrons in beam

ELECTRON MICROSCOPY
 TEM – 1931 – Max Knoll and Ernst Ruska – Nobel prize to Ruska in 1986
 Bulk beam transmitted non scattering electrons
 Electrons pass through thin specimens (50-1000 nm) – OsO4/KMnO4
 2D image on fluorescent screen - Denser regions appear darker
 Resolution – 0.005 nm (Theoretical), 0.05 nm (Practical) ----- 200 nm light
 SEM – 1935 – Max Knoll
 Narrow beam electrons reflected from
the surface of thick metal coated specimens
 Signal sent to cathode ray tube
 Resolution – < 7 nm
 3D image like a television picture
 Lower magnification than TEM

ELECTRON MICROSCOPY
 Confocal SEM – Laser beam illuminates spots on the specimen
 Analytical EM – Elemental composition of materials within tissues
 STEM – 1983 von Ardenne, resolution 0.05 nm
 Immunoelectron microscopy – TEM/SEM imaging of specimens labelled
with gold particles (1-40 nm) conjugated to primary Ab against target Ag
 Visualisation of Ag within ultrastructural images

Staphylococcus Streptococcus Strep pneumoniae
E coli Pseudomonas Vibrio cholerae
Helicobacter Capsule RBCs

Rotavirus under IEM Influenza virus Bacteriophage
Adenovirus Release of viral progeny Mitochondria
Staph epidermidis biofilm Cytopathic effects Golgi body

Aspergillus, sporangium surrounded by hyphae
Influenza Decondensation of chromatin Silicon atoms

 1985
 Electron beam replaced by positively charged Gallium ions
 Liquid metal coated Tungsten needle as ion source
 Cross sectional imaging possible through sputtering
FOCUSSED ION BEAM MICROSCOPY
DUAL BEAM MICROSCOPY
 SEM + FIB Microscopy
 High resolution
 Suitable for fragile specimens

 Uses variety of different interactions of a fine tip with the specimen
 Image created by physical contact of probe moving across the specimen
 Piezoelectric elements facilitate precise movements
 Carbon nanotube probes held at atom’s diameter from sample
 Electrons tunnel between the tip and specimen, producing a signal
 Living objects can be examined
 Scanning Tunneling Microscope
 1981: Binnig and Rohrer – Noble prize 1986
 Resolution - 0.1 nm lateral, 0.01 nm depth
 Used in ultra high vacuum, air, liquid or gas
 Temp ranging from near zero Kelvin to a few hundred degrees Celsius
 Probe moves up and down on the image with steady tunneling current
 Variation in current is detected to create image
SCANNING PROBE MICROSCOPY

SCANNING PROBE MICROSCOPY
 Atomic Force Microscope
 1986: Binnig, Quate and Gerbe
 Resolution: 5 nm (lateral), 0.01 nm (depth), higher in air
 Sharp probe moves over specimen surface at constant distance
 Up and down movement of probe is detected to create true 3D image
 No specimen preparation required
 Cell membrane, flagella, protein, nucleic acid, secretions, DNA sequencing
 Tapping mode and lift mode AFM
 Limitations
 Low magnification
 Cannot delineate steep walls or overhang
 Images affected by choice of tip
 Piezoelectric limitations

TOTAL INTERNAL REFLECTION MICROSCOPY
 Designed for measurements of surface diffusion in biosensors
 DC power supply for electrophoretic flow characterization
 Receptor ligand interactions at nanoscale
OPTICAL NANOSCOPY
 Designed for measurements of surface diffusion in biosensors
 DC power supply for electrophoretic flow characterization
 Receptor ligand interactions at nanoscale

MICROARRAY/NANOARRAY
 Microarray - High-throughput analysis of biomolecules
 Requires large sample volume, prolonged incubation time
 Bulky instrument for detection, laborious procedure
 Nanoarray – Higher sensitivity, simple sample preparation
 Biomolecular analysis
 Monitoring of trace pathogens
 Nucleic acid, enzyme, protein detection using AFM
SURFACE ENHANCED RAMAN SPECTROSCOPY
 Identifies even single molecule based on its vibrational energy
 Rapid DNA sequencing
 Pathogen detection in clinical and environmental samples
 Nanostructure characterization

TYPES OF NANOMATERIALS
 Unidimensional – Surface coatings, engineered surfaces, thin films
 Kaolinite derived aluminosilicate nanoparticle infusion in traditional gauze
 Water absorbing zeolite that concentrates coagulation factors to stop bleeding
 Bioceramic materials
 Bioinert Nanoalumina implant
 Bioresorbable Hydroxyapatite/tricalcium phosphate coating on metallic orthopaedic implant
 Bioactive Bioglass/Apatite wollastonite glass
Nanofunctionalized zirconia and bone cement additives – Synthetic bone
 Bidimensional – Carbon nanotubes, nanowires, biopolymers
 Tridimensional – Quantum dots, dendrimers
 Plasmid DNA expression vectors in wounds to enhance growth factors
 Electroporation mediated transfection
 Starbust dendrimers get endocytosed and release DNA to nucleus
 Polyamidoamine (PANAM) dendrimer for in vitro gene delivery – Hypoxic injury
 Nanoengineering of stem cells to make organs

BIOLOGICAL NANOPARTICLES
 Proteins, enzymes, peptides, DNA, RNA
 Genetically engineered fluorescent viruses identify E. coli by infection
 Fluorescent microscope detect glow in a few hours
 Use of bacteria to transport ‘smart nanoparticles’ to specific targets
 Precise position of sensors within cells
 Drug/DNA delivery
 Diagnosis and treatment of diseases
 Carbon nanotubes delivered to target,
heated, selective killing of diseased cells

 Polymers
 Gelatin, albumin
 Polyethylene gycol, polylactide,
polyepsilon caprolactone, polyalkylcyanoacrylate
 Porous silicon
 Carbon nanotubes
 Carbon nanospheres
 Dendrimers <15 nm
 Deliver DNA in gene therapy
 Nanogold
 Nanoscale sensors and actuators
 Respirators
 Protective clothing – Nanobionic in hypothermia
SYNTHETIC NANOMATERIALS

 Nanowires – Lateral dimension 1-100 nm
SYNTHETIC NANOMATERIALS

QUANTUM DOTS
 Combination of microfluidics, magnetic particles and gold nanoparticles
encoded with antibodies and DNA
 Extremely high sensitivity
 Electrochemical immunoassay - IgG
 Detect individual biomolecules and virus particles

SYNTHETIC NANOMATERIALS: BIOSENSORS
 EM
 LF-ICT
 Pathogen detection
 Screen for disease markers (Infections, cancer)
 Fluorescent organic dye attached to Salmonella Ab
 Targeted/controlled drug therapy
 Magnetic nanoparticles
 Stealth nanoparticles - PEG – Least opsonization on the surface in vivo
 Least uptake by macrophages – Persistence in blood
 Silver nanocrystals for antimicrobial wound dressing
 Releases clusters of highly reactive silver cations upto 100 ppm on contact with water
 Causes electron transport , cell membrane damage, inactivation of bacterial DNA, binding
of insoluble complexes in microbes
 Nanoparticle cream for delivery of nitric oxide to treat infection
 Drug delivery across blood brain barrier
 Nanosphere liposomes, nanocapsules

 Biomolecular interactions
 Immobilization of ssDNA on cantilever
 Electrochemical detection of DNA hybridization
 DNA sequencing using nanoprobes
 Identification of molecular recognition
 Identification of self assembly motifs
 MWNT biosensors for detection
 Hydrazine in hypertension
 Epinephrine and dopamine in Parkinsonism
 Glucose in diabetics
 Cholesterol, uric acid
 Hemin modified sensors for oxygen, NADPH, H2O2, NO
 Organophosphates

 Imaging technologies – Qdot nanocrystals
 Live cell imaging and dynamics - Multicolour analysis
 Permanent sample storage in pathology
 Environmental monitoring
 Interferometric sensor
 Binding of virus particle to Ag specific (haemagglutinin) Ab
 Handheld device for detection and quantification of virus
 Rapid screening of pathogens from samples in outbreaks

EMERGING NANO-CONCEPTS
 Rapidly Adaptable Nanotherapeutics
 Inhaled nanoparticles loaded with siRNA
 Can target and shutdown specific genes
 Specific microbes can be targeted
 Successful in HCV
 Being tested against bacteria
 Possible use against bioweapons
 DNA vaccines for bacteria, viruses, parasites
 Silver ions + oxygen can destroy HIV
 Peptide nanoparticles overcome resistance

EMERGING NANO-CONCEPTS
 Pharmacy on a chip
 Nanorobots – Mobile ATP energized nanomachines
 Respirocyte (1μ RBC) – 10000 times more efficient than RBC
 Microbivore (3.4μ X 2μ Phagocyte)
 Ingested bacteria converted to aa, ffa, sugar, nucleotides
 Nanomimicry
 Nanorubber - Self healing rubber
 Artificial tissues and organs
 Nanopistons (Incorporation of H2, O2, N2 in nanotubes)
 Nanomaterials can trick bacteria to sense a quorum early when there are
few bacteria. This will prompt natural immunity to overcome them
 Force spectroscopy to manipulate single membrane proteins
 Mapping of surface properties and receptor sites
 Measurement of cellular interactions at single cell/ molecule level

FUTURE
 Artificial blood
 Nanorobotics - Respirocyte (1μ RBC)
 10000 times more efficient than RBC

NANOTECHNOLOGY ISSUES
 Ethical, legal and social aspects
 No long-term experience
 Few exposure assessments
 Toxicity: Few toxicological assessments
 Removal difficult
 Pollution
 Entry into food chain
 Stem cell research
 Self replicating machines

Nanotechnology and Infectious Diseases

Nanotechnology and Infectious Diseases

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