Nanotechnologies in Biosafety Nanotechnologies in Biosafety Applications Applications

Jeremy TzengJeremy TzengBiological SciencesBiological SciencesClemson UniversityClemson University

May. 14, 2007

Topic AreasTopic Areas

AdhesinAdhesin--Specific Nanomaterials in Biosafety Specific Nanomaterials in Biosafety ApplicationsApplications

Physical Removal of PathogensPhysical Removal of PathogensBiosensor ApplicationsBiosensor Applications

TiOTiO22--Based SelfBased Self--Disinfecting SurfaceDisinfecting Surface

ABC NewsABC News

FDA Bans Use of FDA Bans Use of BaytrilBaytril in Poultryin PoultryThursday July 28, 2005 8:53pmThursday July 28, 2005 8:53pm

Washington (AP) Washington (AP) -- The Food and Drug The Food and Drug Administration is banning the use of the Administration is banning the use of the antibiotic antibiotic BaytrilBaytril in poultry because of in poultry because of concerns the drug could lead to antibioticconcerns the drug could lead to antibiotic--resistant infections in people.resistant infections in people.

Problems and ApproachProblems and Approach

Problems associated with the use of Problems associated with the use of antibioticsantibiotics

Emerging multiple antibiotic resistant bacteria Emerging multiple antibiotic resistant bacteria due to mutations/recombinationdue to mutations/recombinationUndesirable side effectsUndesirable side effectsIncreased costs for the development of new Increased costs for the development of new antibioticsantibiotics

ApproachApproachDevelopment of alternative nonDevelopment of alternative non--antibiotic antibiotic treatments for specific pathogenstreatments for specific pathogens

Copyright: SigmaCopyright: Sigma--AldrichAldrich

HostHost--Pathogen InteractionsPathogen Interactions

Earliest events in bacterial infectionsEarliest events in bacterial infectionsRequired for Required for extracellularextracellular colonization or colonization or internalizationinternalizationColonization mediated by bacterial adhesinsColonization mediated by bacterial adhesinsAdhesins recognize and bind to specific receptor Adhesins recognize and bind to specific receptor moieties of host cellsmoieties of host cellsReceptor binding activates new genes important Receptor binding activates new genes important in the pathogenic processin the pathogenic process

Blocking of interactions Blocking of interactions --> effective treatment> effective treatment

Bacterial AdhesinsBacterial Adhesins

HairHair--like appendages called like appendages called pilipili or or fimbriaefimbriaethat extend out from the bacterial surfacethat extend out from the bacterial surfaceOr, directly associated with bacterial Or, directly associated with bacterial surface surface –– nonpilusnonpilus adhesins. e.g. adhesins. e.g. intiminintimin of of E. coli E. coli O157:H7O157:H7

Bacterial Binding to Host Mediated by Adhesins

Bacterial Adhesins

Lefkowitz S., et. al., 2000, Glycoscience

Transmission electron micrograph of E. coli adhering to epithelium in the intestine of a pig.

Moon, H.W. 1997. Comparative histopathology of intestinal infections. In: Mechanisms in the pathogenesis of enteric diseases (P.S. Paul, D.H. Francis and D.A. Benfield, eds.) Adv. Exptl. Med. Biol. 412:1. Plenum Press, New York.

Copyright © Gary E. Kaiser

Examples of Bacterial Adhesins and their ReceptorsExamples of Bacterial Adhesins and their Receptors

Sharon, N., and I. Ofek, Glycoconj. J. 17, 659-664 (2000)

Organism Adhesin Receptor E. coli Type 1 pili (FimH) D-mannose Intimin Tir Campylobacter jejuni CadF Fibronectin

E. coli ORN178(FimH+, GFP)

E. coli ORN208(FimH-, GFP)

ObjectivesObjectives

Development of carbohydrate Development of carbohydrate biofunctionalized nanoparticles (biobiofunctionalized nanoparticles (bio--NP) NP) that have affinity for pathogen adhesinsthat have affinity for pathogen adhesinsEvaluation of these synthesized bioEvaluation of these synthesized bio--NP NP (mimicking host cell surface receptors) for (mimicking host cell surface receptors) for their binding to pathogenstheir binding to pathogensDevelopment of alternative nonDevelopment of alternative non--antibiotic antibiotic treatment for bacterial infectionstreatment for bacterial infections

ApproachesApproaches

Multivalent nanoparticles specific to Multivalent nanoparticles specific to adhesins of targeted pathogens adhesins of targeted pathogens Aggregation of pathogens mediated by Aggregation of pathogens mediated by nanoparticles nanoparticles –– prevents binding to host prevents binding to host cellscells

Nanoparticle-Bacterial Bindings

High NP Concentration:Bacterial Isolation

Intermediate NP Concentration:Bacterial Agglutination

Low NP Concentration:Bacterial Tagging

Bacterial cellNanoparticles

Nanoparticle Chemical Structure: Mannose Functionalization

Polystyrene corePolyethylene glycol tether

Mannose asBiofunctional group

Nanoparticle Design Strategy

Functionalized PEG side chains extending from hydrophobic polymer backbone chain. Diagram illustrates the self assembly into the nanoparticles followed by photochemical curing.

TEM/SEM TEM/SEM Images of Images of

NanoparticlesNanoparticles

200 nm

Journal ofBiomedical Nanotechnology

Vol.1, 61–67, 2005

E. coli - NP Interaction

a b

dc

e f

TEM images showing the agglutination of E. coliORN178 mediated by D-mannose-tethered nanoparticles

(a,b) Lower magnification and (c,d) higher magnification

(e) E. coli ORN178 only (similarly with bare nanoparticles)

(f) E. coli ORN208 with the same D-mannose-tethered polymeric nanoparticles.

Journal of Nanoscience and Nanotechnology, Vol.5, 319–322, 2005

Aggregation Mediated by NPsAggregation Mediated by NPs

ORN178 with Man-NPs ORN178 with Man-NPs

ORN208 with Man-NPs ORN178 with Gal-NPs

Journal of Biomedical Nanotechnology, Vol.1, 1–6, 2005

CFU Reduction of CFU Reduction of E. coli E. coli ORN178ORN178

Journal of Biomedical NanotechnologyVol.1, 1–6, 2005

AdvantagesAdvantages

Low costLow costPolystyrene corePolystyrene coreSimple carbohydrate Simple carbohydrate biofunctionalbiofunctional groupsgroupsEasy transportation and storage conditionEasy transportation and storage condition

Neither antibiotic nor antibody approachNeither antibiotic nor antibody approachLess prone to the effects of mutations vs. an Less prone to the effects of mutations vs. an antibiotic approachantibiotic approach

Mutation in adhesin Mutation in adhesin –– lost of ability to lost of ability to attach to host cellsattach to host cells

Microarray for the Screening of Microarray for the Screening of Receptor MoleculesReceptor Molecules

ApplicationsApplications

Purging of enteropathogens from intestinal Purging of enteropathogens from intestinal tracks of commercial poults to allow for tracks of commercial poults to allow for establishment of beneficial micro floraestablishment of beneficial micro floraPurging of enteropathogens from intestinal Purging of enteropathogens from intestinal tracks of commercial poultries prior to their tracks of commercial poultries prior to their processprocessDisplacing technologyDisplacing technology

Non antibiotic approach for the prevention and Non antibiotic approach for the prevention and treatment of infectious diseasestreatment of infectious diseasesAdhesinAdhesin--specific drug deliveryspecific drug delivery

Diagnostic applicationsDiagnostic applications

Adhesin in Biosensor ApplicationsAdhesin in Biosensor Applications

Current target capturing molecules used in Current target capturing molecules used in biosensor applications:biosensor applications:

DNA or RNA sequencesDNA or RNA sequencesProteins or antibodiesProteins or antibodies

Highly specificHighly specificHigh costHigh costShort shelf lifeShort shelf lifeRequires special instrumentRequires special instrumentAvailabilities of specific antibodiesAvailabilities of specific antibodiesAntibodies showing cross reactionsAntibodies showing cross reactions

Adhesin in Biosensor ApplicationsAdhesin in Biosensor Applications

Fabrication of microchip surface with Fabrication of microchip surface with receptor molecules specific for targeted receptor molecules specific for targeted microorganisms.microorganisms.Binding of microorganisms to microchip Binding of microorganisms to microchip surface could cause changes in weight, surface could cause changes in weight, conductivities, or resonance frequencies conductivities, or resonance frequencies ––realreal--time biosensor for food safety time biosensor for food safety applicationsapplications

Intellectual PropertyIntellectual Property

US patent application entitled US patent application entitled ““AdhesinAdhesin--Specific Nanoparticles and Process for Specific Nanoparticles and Process for Using SameUsing Same”” filed in 2003.filed in 2003.

AcknowledgementsAcknowledgementsFunding:Funding:

USDAUSDAUndergraduate Research, Discovery, and Creative Activity Undergraduate Research, Discovery, and Creative Activity Initiative, Clemson UniversityInitiative, Clemson UniversitySCSC--LIFE/HHMILIFE/HHMI

Collaborators:Collaborators:Clemson UniversityClemson University

R. Latour, Dept. of BioengineeringR. Latour, Dept. of BioengineeringF.J. Stutzenberger, Dept. of Biological SciencesF.J. Stutzenberger, Dept. of Biological SciencesY.Y.--P. Sun, Dept. of ChemistryP. Sun, Dept. of ChemistryGeorge Luo, Dept. of Biological SciencesGeorge Luo, Dept. of Biological Sciences

North Carolina State UniversityNorth Carolina State UniversityJesse Grimes, Dept. of Poultry ScienceJesse Grimes, Dept. of Poultry Science

University of TennesseeUniversity of TennesseeJun Lin, Dept. of Animal ScienceJun Lin, Dept. of Animal Science

TiOTiO22--Based SelfBased Self--Sterilizing SurfaceSterilizing Surface

Titanium dioxide also known as:Titanium dioxide also known as:Titanium oxide or Titanium oxide or titaniatitania

Exists in a number of crystalline formsExists in a number of crystalline formsRutileRutileAnataseAnataseBrookiteBrookite

Molecular mass: 79.87 g/mol Molecular mass: 79.87 g/mol Density: 4.23 g/cmDensity: 4.23 g/cm33

Melting point: 1870 Melting point: 1870 °°C (3398 C (3398 °°F) F) Boiling point: 2972 Boiling point: 2972 °°C (5381.6 C (5381.6 °°F)F)

Current Applications of TiOCurrent Applications of TiO22Pigment for providing whiteness and opacityPigment for providing whiteness and opacity

Paints, coatings, plastic, paper, inks etc.Paints, coatings, plastic, paper, inks etc.High refractive indexHigh refractive index

Low levels required for a white opaque coatingLow levels required for a white opaque coatingEffective opacifier for pigmentsEffective opacifier for pigmentsUsed as an opacifier in glass, enamels, cosmetics, sunscreens etUsed as an opacifier in glass, enamels, cosmetics, sunscreens etc.c.

Fog proof, and self cleaning glassFog proof, and self cleaning glassSunscreensSunscreens

TiOTiO22ZnOZnO

AntiAnti--bacterial, antibacterial, anti--viral, fungicidalviral, fungicidalDeodorizing, air purificationDeodorizing, air purificationWater treatment, water purificationWater treatment, water purification

TiOTiO22 as a Photocatalystas a Photocatalyst1.1. The photoexcited TiOThe photoexcited TiO22 catalyst produces electroncatalyst produces electron--hole pairs that migrate hole pairs that migrate

to the TiOto the TiO22 surface.surface.2.2. The photogenerated holes in TiOThe photogenerated holes in TiO22 can react with adsorbed Hcan react with adsorbed H22O or OHO or OH-- at at

the interface to produce highly reactive hydroxyl radicals.the interface to produce highly reactive hydroxyl radicals.3.3. The electrons react with OThe electrons react with O22 to form superoxide ions.to form superoxide ions.4.4. Active radicals generated can oxidize cells on the TiOActive radicals generated can oxidize cells on the TiO22 surface.surface.

Phases of TiOPhases of TiO22

AnataseAnatase::OctahedriteOctahedrite, a tetragonal mineral of dipyramidal habit, a tetragonal mineral of dipyramidal habitPhotocatalyticallyPhotocatalytically active under UV lightactive under UV light

RutileRutile::Tetragonal mineral usually of prismatic habit, often twinnedTetragonal mineral usually of prismatic habit, often twinnedStable under ambient conditionsStable under ambient conditions

BrookiteBrookite::OrthorhombicOrthorhombicDifficult to prepare due to high purity and large surface areaDifficult to prepare due to high purity and large surface areaHydrothermal conditions are necessary for productionHydrothermal conditions are necessary for production

TiOTiO22 Band GapBand Gap

Brookite type

AnataseAnatase: 3.2 : 3.2 eVeVRutileRutile: 3.02 : 3.02 eVeVBrookiteBrookite: 2.96 : 2.96 eVeV

S. Banerjee, 2006S. Banerjee, 2006

Antimicrobial AssayAntimicrobial AssayTiOTiO22: (10, 1, 0.1, or 0.01 mg/ml): (10, 1, 0.1, or 0.01 mg/ml)

Commercial Commercial titaniatitania P25 (79% P25 (79% anataseanatase and 21% and 21% rutilerutile))BrookiteBrookite

UV:UV:Wavelength: 365 nmWavelength: 365 nmIntensity: 350 to 360 Intensity: 350 to 360 µµw/cmw/cm22..At this intensity UV alone has little or no effect on bacterial At this intensity UV alone has little or no effect on bacterial growthgrowth

101088 E. coliE. coli cells were washed with phosphate buffered saline cells were washed with phosphate buffered saline (PBS) and transferred onto Petri dishes containing 18 ml of (PBS) and transferred onto Petri dishes containing 18 ml of TiOTiO22..The samples were exposed to UV light with constant mixing for The samples were exposed to UV light with constant mixing for 120 minutes and plated every 30 minutes.120 minutes and plated every 30 minutes.Viable cell counts were obtained.Viable cell counts were obtained.

Results: P25 Anatase SampleResults: P25 Anatase SampleAnatase 1MG/ML

0.E+00

1.E+05

2.E+05

3.E+05

4.E+05

5.E+05

6.E+05

0 30 60 90 120

TIME (SECONDS)

CO

LON

Y FO

RM

ING

U

TIO2 W/UV

TIO2 W/OUT UV

Anatase 1MG/ML

1.E+00

1.E+01

1.E+02

1.E+03

1.E+04

1.E+05

1.E+06

1.E+07

0 30 60 90 120

TIME (SECONDS)

CO

LON

Y FO

RM

ING

U

TIO2 W/UV

TIO2 W/OUT UV

DH2O W/UV

DH20 W/OUT UV

• TiO2 nanoparticles caused a 5-log reduction in viable cells after 30 minutes of exposure.

• In the absence of UV light, TiO2 nanoparticles only exhibited a 1-log reduction after 60 minutes and a 3-log reduction after 120 minutes of exposure.

• UV light alone did not exhibit significant cell death.• The control cells did not exhibit any reduction after 120 minutes.

Results: CU Brookite SampleResults: CU Brookite SampleBrookite 1MG/ML

0.E+00

1.E+05

2.E+05

3.E+05

4.E+05

5.E+05

6.E+05

7.E+05

0 30 60 90 120

TIME (SECONDS)

CO

LON

Y FO

RM

ING

U

TIO2 W/UV

TIO2 W/OUT UV

Brookite 1MG/ML

1.E+00

1.E+01

1.E+02

1.E+03

1.E+04

1.E+05

1.E+06

1.E+07

0 30 60 90 120

TIME (SECONDS)

CO

LON

Y FO

RM

ING

U

TIO2 W/UV

TIO2 W/OUT UV

DH2O W/UV

DH20 W/OUT UV

• TiO2 nanoparticles exhibited a 5-log reduction after 30 minutes of exposure. • In the absence of UV light, TiO2 nanoparticles only exhibited a 1-log reduction after

60 minutes and a 4-log reduction after 120 minutes of exposure.• UV light alone did not exhibit significant cell death.• The control cells did not exhibit any reduction after 120 minutes.

SEM Observations of TiOSEM Observations of TiO22 Induced Cell DamagesInduced Cell Damages

Cell Damages:Cell Damages:Loss of cell envelopeLoss of cell envelopeProtuberances in the cell envelopeProtuberances in the cell envelopeCell enlargementCell enlargementCell surface depressionsCell surface depressions

SEM Images of SEM Images of E. coliE. coli

Experimental:Experimental:Cells exposed under UV light Cells exposed under UV light

for 30 minutes to Brookite TiOfor 30 minutes to Brookite TiO22

Control: Control: Untreated cellsUntreated cells

SEM Images of SEM Images of E. coliE. coli

Experimental:Experimental:Cells exposed under UV light Cells exposed under UV light

for 30 minutes to Brookite TiOfor 30 minutes to Brookite TiO22

Control: Control: Untreated cellsUntreated cells

Band Gap ReductionBand Gap ReductionNanoNano--sized particlessized particles

Better physical and chemical properties in comparison to Better physical and chemical properties in comparison to bulk materials.bulk materials.The large surface area promotes better interaction between The large surface area promotes better interaction between the devices and other media in contact with the surface. the devices and other media in contact with the surface. Chemically stableChemically stable

Toxicity data limitedToxicity data limited

High absorption in the UV light regionHigh absorption in the UV light region

Doped TiODoped TiO22Metals: FeMetals: Fe3+3+, Mo, Mo5+5+, Rh, Rh3+3+, V, V4+4+, and Ru, and Ru3+3+

Expensive ionExpensive ion--implantation facilitiesimplantation facilitiesUnstableUnstable

NonNon--metals: N, F, S, C, I, P, B and Hmetals: N, F, S, C, I, P, B and H

VisibleVisible--LightLight--Activation of TiOActivation of TiO22

VLA SelfVLA Self--Disinfecting SurfaceDisinfecting SurfaceCoating of indoor surfacesCoating of indoor surfaces

Walls, floors, appliances, and etc.Walls, floors, appliances, and etc.Food preparation surfacesFood preparation surfaces

Antimicrobial activities remain to be Antimicrobial activities remain to be quantifiedquantifiedSafety data Safety data

TiOTiO22 Project AcknowledgementsProject AcknowledgementsDepartment of Biological SciencesDepartment of Biological Sciences

Rupal ShahRupal ShahDonna WeinbrennerDonna Weinbrenner

Clemson Microscopy Imaging FacilityClemson Microscopy Imaging FacilityMr. Darryl KruegerMr. Darryl KruegerMr. Dayton CashMr. Dayton CashMr. Donald MulweeMr. Donald MulweeMr. Amar KumbharMr. Amar KumbharDr. Joan HudsonDr. Joan Hudson

Department of Materials Science & EngineeringDepartment of Materials Science & EngineeringDr. Burt LeeDr. Burt LeeRadhika BhaveRadhika BhaveSujaree KaewgunSujaree KaewgunChris Chris NolphNolph