Hans J. Griesser

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Ethics in nanotechnology research case studies

• Hans J. Griesser
• Ian Wark Research Institute
• University of South Australia
• CAPPE workshop,
• ANU
• 22 July 2009

Eremophila splendens
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Ethics issues in nanotech research at the Wark

• The Warks research is on minerals and materials
  (polymers)
• Minerals research has not presented specific
  ethics issues.
• Ethics comes in mostly in our work on
  materials/devices for human health applications
• Nanoparticles for cancer diagnostics and
  treatment
• Antibacterial silver nanoparticles
• Ethical, moral and legal issues arising from
  intellectual contributions to research by
  knowledge provided by traditional Australian
  Aboriginal peoples
• And one needs to keep an eye out for general
  research ethics
• thou shalt not exaggerate claims, novelty,
  applicability etc
  revolutionary breakthrough. , have made
  the startling discovery that. , will
  revolutionise usage in technological fields such
  as xxx

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Case study Nanoparticles for cancer diagnostics
and treatment

• Superparamagnetic Nanoparticles give a negative
  contrast in MRI, enable magnetic manipulation (lt
  respond to strong magnet) and produce heat in
  response to external alternating magnetic fields.
• Improved detection of smaller tumours.

(B. Thierry, UniSA)
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• Nanoparticles for the delivery of anti-cancer
  drugs
• Injected into blood circulation
• Size matters

(Chris Barbe, ANSTO)
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• In both cases, there are two important issues
• Evasion of recognition by the immune system
• Specific targeting to tumour cell surfaces

(B. Thierry, UniSA)
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Ethics issues

• Animal ethics approval is needed for Phase I
  clinical trials
• Rules and procedures are well established
• BUT
• Ethics committees know about surgical trials,
  know little about nanotechnology / materials
• How confident do we want to be, and how confident
  can we be, that we have done a thorough set of
  lab trials to make sure that the animals die for
  a good cause ?
• In vitro model experiments can be poor
  predictors of in vivo. How much time do we want
  to invest to develop better models ?
• Judgement call more results or faster progress
  ?
• Cancer research is a hot topic might lose out
  on first publication
• Have the clinical experiments been well planned
  from the materials perspective ?

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Infection on biomedical devices and implants

• Clinical problem infections at biomedical
  devices and implants
• Affects the lives of patients and causes enormous
  costs to health care systems
• Urinary catheters almost 100 within 7 days
• Percutaneous catheters
• Hip and knee implants 3-4
• Main cause Staphylococcus species (S. aureus
  and S. epidermidis)
• Infection can arise many months after surgery
• Contact lenses rare in first world, easy to
  remove
• Etc

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Antibacterial coatings

• Can a coating prevent bacterial biofilm formation
  ?
• Probably no universal recipe, technical
  specifications vary with the device/implant
• For catheters, weeks for implants, years
• Needs to meet other criteria too, eg tissue
  compatibility and integration for hip and knee
  implants
• We need to test effects of coatings on mammalian
  cells and tissue
• We need to consider effects on remote organs if
  antibacterial compounds are released

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Case study silver nanoparticles

• Silver has long been known to have antibacterial
  effects
• Silver metal itself appears to be ineffective
  oxidation required, then release of silver ions.
  Or use silver salts
• Silvadene Cream 1 is one of the most widely
  used antibiotic creams for infections associated
  with burns.
• Many studies over last 15 years, several start-up
  companies, mainly in US, selling silver based
  antibacterial coatings or products
• Questions remain over effectiveness as well as
  toxicity to human cells
• Coating strategies for biomedical implants not
  optimal thickness (non-)uniformity, lack of
  control over loading/release, etc

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Our research release silver ions from silver
nanoparticles embedded in ultrathin polymer
coatings

• Key features
• High uniformity of coating
• Control of coating thickness
• Control of loaded amount
• Control of release kinetics via crosslink density
  of polymer or via an additional layer
• Select a polymer that is known to offer good
  support for attachment of human cells and tissue

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Control of release kinetics

• Additional polymer layer controls outdiffusion

6 to 30 nm thick
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Antibacterial effectiveness
Polymer coating - control sample (no silver
nano- Particles)
Polymer coating loaded with Ag nanoparticles
Polymer coating loaded with Ag nanoparticles and
covered with another 6 nm thick polymer layer
Silver loaded coatings are highly effective
against bacteria
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But with mouse fibroblast (skin) cells
Control sample (polymer with Silver loaded
polymer coating no silver nanoparticles)
Cells attach, then die Cells attach and spread
well The antibacterial coatings kill mouse cells
with equal effectiveness !
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Concerns

• Controversy in the literature often researchers
  have not considered silver nanoparticles versus
  silver ions
• Metallic silver nanoparticles may be safe but
  also ineffective against bacteria release of
  silver ions is needed for antibacterial action
• Dose dependent effects but to kill bacteria, we
  need amounts that are toxic to mammalian cells
  too !
• Mechanism of action Ag ions can complex with
  amino acids such as cysteine in proteins, and
  with DNA/RNA in bacteria as well as humans !
• Accumulation of heavy metal ions in higher
  organisms is well known what will happen on
  release of lots of silver ions into the
  environment from silver in socks, washing
  machines, etc ?

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Ethical dilemma

• Silver clearly has a place in modern medicine,
  for example for use in antibacterial cream for
  burn wounds.
• BUT it should be used with caution, only when
  there is no safer option.
• Given the clearly documented toxicity of silver
  ions to human cells and the likelihood of
  eventual accumulation in organisms, how much /
  where / how should a concerned scientist speak
  out against what seems to be misuse of technology
  ?
• Should I say in public that the use of silver in
  socks, washing machines, etc may be dangerous in
  the longer term, and unnecessary anyway there
  are safer solutions (for example, change socks
  more often..).

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Case study Aboriginal knowledge as a factor in
the development of nano-coatings for biomedical
devices and implants

• Traditional medicinal knowledge of Australian
  Aboriginal people extends over a long time period
• No written records from Aboriginal sources
  sketchy records of information transmitted to
  white Australians
• Traditional knowledge often not attributable to
  specific peoples, tribes, owners
• No basis for authorship or ownership as we
  understand it in modern scientific publications
  and intellectual property laws
• Yet, we cannot simply ignore the contributions
  made by such traditional knowledge how do we
  recognise it, honour it, and reward the
  traditional sources of such information ?

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General design of our non-release antibacterial
coatings
Interfacing polymer coating - very thin
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Natural antibacterials

• Australian Aboriginal people used traditional
  medicines as skin ointments and throat washes,
  and also against sores, influenza, eye and ear
  infections which suggests an antibacterially
  active agent
• Two plant species from the genus Eremophila were
  mainly used by Aboriginal people for such
  purposes
• The active ingredients have not been identified
• Literature Compounds extracted by structural
  chemists from various Eremophila species and
  chemical structures identified
• Many highly unusual compounds, esp diterpenes,
  cyanogenic glycosides, verbascides, etc
• None tested for antibacterial effectiveness

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Eremophila plants

• (Eremos desert, lonely place phileo love)
• A large and diverse genus, 215 scientifically
  named species
• Uniquely Australian
• Most species grow in remote arid areas. Often
  limited range.
• Only a few species used in traditional Australian
  Aboriginal medicine

E glabra
E gilesii
E splendens
E ovata
(all photos H Griesser)
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Research questions

• Why are only 4 of 215 species known to have been
  used for antibacterial purposes ?
• Probable answers
• Distribution those 4 species grow over much of
  inland Australia useful for nomadic peoples
• Knowledge of use of others may have become
  extinct, especially in Western Australia
• Are extracts from other Eremophila species also
  active ?
• What are the antibacterial chemicals ?
• Can we use them as coatings
• to protect biomedical devices ?

E duttonii
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Some are hairy. and inactive
Eremophila margarethae Eremophila
warnesii Eremophila glabra form
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.. and some are very sticky . and
antibacterially active !
E gibbosa

• E alternifolia E platythamnos
  E sp nov aff gibbosa?

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Identification of active compounds

• Extraction of resin with diethyl ether
• Activity-guided fractionation, HPLC, column
  chromatography
• Electrospray MS molecular weights
• NMR determination of molecular structures
• Optical rotation chiral or racemic ?

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Antibacterials from Eremophila serrulata
1 general structure of serrulatane Antibacterial
activity 2 gt 4 gt 3 gt 5. Active against
Gram-positive bacteria including MRSA
Staphylococcus aureus, Streptococcus pyogenes,
and Streptococcus pneumoniae. Minimum inhibitory
concentrations 1.95 to 31.3 mg/mL minimum
bactericidal concentrations 3.91 to 62.5
mg/mL. No activity against Gram-negative
bacteria.
C.P. Ndi, S.J. Semple, H.J. Griesser, S.M. Pyke,
M.D. Barton Phytochemistry, 68, 2684-2690 (2007).
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ToF-SIMS Analysis
Ald-pp PAA SER
Ald-pp PAA
Parent ion (PA) spectral region PA
C20H28NO2, m/z 314.21
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Bacterial testing of amide-coupled
carboxy-serrulat-ene, on PAA/aldehyde pp

• 2-hour attachment assay gt 99 reduction !
• Remaining very few bacteria on coating defect
  spots ?

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Longer growth times

• 6 hrs incubation in bacterial growth solution
• S. epidermidis
• Biofilm formed on PAA still no bacteria on PAA
  SER

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3T3 fibroblast cell attachment on serrulatane
coated samples
24 hours 48 hours
PAA (control)
SER 0.015
SER 0.0075

• Surface density matters

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Back to the ethics..

• Traditional knowledge not attributable to
  specific peoples, tribes, owners
• No basis for authorship or ownership as we
  understand it in modern scientific publications
  and intellectual property laws
• Yet, we cannot simply ignore the contributions
  made by such traditional knowledge how do we
  recognise it, honour it, and reward the
  traditional sources of such information ?
• Precedents yew, echinacea, quinine,
• One idea set up organic production of
  serrulatanes, via plantations of Eremophila
  plants in Aboriginal communities
• What else could we do to reward traditional
  knowledge ?

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THE END Thank you !
Here you can find Eremophilas
Uluru (Ayers Rock), Central Australia
E macdonnellii