NANOTECHNOLOGY IN INFECTIOUS DISEASES Major Inam Danish Khan

Dept of Microbiology and Molecular MedicineArmy 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

http://en.wikipedia.org/wiki/File:ScanningTunnelingMicroscope_schematic.png

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

SYNTHETIC NANOMATERIALS: BIOSENSORS 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

SYNTHETIC NANOMATERIALS: BIOSENSORS 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

LAB ON A CHIP

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

THANK YOU