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L’Hocine YAHIA
Laboratoire d'innovation et d'analyse de bioperformances
1
New Developments
in Sterilization and Decontamination
using Nanotechnologies
Biomedical Engineering Institute
2014 World Sterilization Congress of the WFHSS
INFECTIONS RELATED TO IMPLANTS
Introduction
Different sources of the infectious agent, such as:
A contaminated implant/device surface
The hands of the surgical staff during implantation/application
The patient’s own skin or mucus membrane
Distant local infections in the patient
Contaminated disinfectants Contact with other patients in the hospital, or family members after
intervention
http://www.google.ca/url?sa=i&rct=j&q=&esrc=s&frm=1&source=images&cd=&cad=rja&docid=WZ77S3RnJHDzwM&tbnid=pKHnRypaatoCZM:&ved=0CAUQjRw&url=http://www.tigran.se/en/professional/application-areas/implantology/peri-implantitis/debridement-at-peri-implantitis/&ei=c0wvUuL6JefX2QWikYGACg&bvm=bv.51773540,d.aWc&psig=AFQjCNFz_65nKzsRmDG1TSclk7tWFMsf5Q&ust=1378917807351674http://www.google.ca/url?sa=i&rct=j&q=&esrc=s&frm=1&source=images&cd=&cad=rja&docid=Hl61oQjFkx4xJM&tbnid=22NOtIvCe3dkYM:&ved=0CAUQjRw&url=http://www.hpc.org.ar/v2/v_art_rev.asp?id=15&offset=21&ei=4k4vUuXmDabZ2wXdhYHICA&bvm=bv.51773540,d.aWc&psig=AFQjCNEX0M689wdQKbL2NuiWZWHSgwRcnw&ust=1378918350288263http://www.google.ca/url?sa=i&rct=j&q=&esrc=s&frm=1&source=images&cd=&cad=rja&docid=VBgn44yF8_4NgM&tbnid=cO5QEoc_3Tl7-M:&ved=0CAUQjRw&url=http://www.boloncol.com/boletin-24/cuidados-de-la-piel-en-el-paciente-oncologico.html&ei=CVEvUqLcDcTr2QXn54GYBg&bvm=bv.51773540,d.aWc&psig=AFQjCNEX0M689wdQKbL2NuiWZWHSgwRcnw&ust=1378918350288263http://www.google.ca/url?sa=i&rct=j&q=&esrc=s&frm=1&source=images&cd=&cad=rja&docid=vzotV1gbnvNVhM&tbnid=kRojBe1wMCVuSM:&ved=0CAUQjRw&url=http://sistemainterno.com/web/comvalsa/category/sklar-instrumental-quirurgico/&ei=w1IvUsSCG-bQ2wWa24GgDQ&bvm=bv.51773540,d.aWc&psig=AFQjCNE1d0f07bbMBBiesCdITfhffSTv_A&ust=1378919447681366http://www.google.ca/url?sa=i&rct=j&q=&esrc=s&frm=1&source=images&cd=&cad=rja&docid=I_B2VPttWwv0wM&tbnid=7ZSY8qfXkA1DvM:&ved=0CAUQjRw&url=http://www.farmalta.com/blog/?paged=2&ei=PlMvUqjFAer12wWx-YGADg&bvm=bv.51773540,d.aWc&psig=AFQjCNE1d0f07bbMBBiesCdITfhffSTv_A&ust=1378919447681366http://www.google.ca/url?sa=i&rct=j&q=&esrc=s&frm=1&source=images&cd=&cad=rja&docid=hLJIrQ878gUwIM&tbnid=847Qtuo5TCCEIM:&ved=0CAUQjRw&url=http://www.rpp.com.pe/2013-02-04-inician-visita-de-hospitales-para-verificar-buen-servicio-a-pacientes-noticia_564066.html&ei=MlUvUpiyO8m52wXPyoCQBA&bvm=bv.51773540,d.aWc&psig=AFQjCNF6OOR5y800dButqHl2DVkkFYWqCg&ust=1378919776783026
PRINCIPAL BACTERIA ON IMPLANTS Introduction
Infections caused by: • Staphylococcus epidermidis • Staphylococcus aureus
70–90% of the implant related infections
IMPLANT BACTERIA
Catheter-related infections Staphylococcus aureus and Staphylococcus epidermidis
Orthopedic implants Staphylococcu aureus
E. coli , Enterobacteriaciae, P. aeruginosa
Lenses S. epidermidis
Skin grafts S. aureus and S. epidermitis
Cardiac valve S. epidermidis , S. aereus, E. faecelis, P. aeruginosa, and Candida albicans
Table 1. Inscidence of bacteria present after different surgery implants
BIOFILM FORMATION
8
Introduction
Fig. 2 Schematic representation of biofilm formation.
Lerberg
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Nanomaterials
Copper et Copper Oxide NPs
Antibacterial material
Iron Oxide NPs (SPIONs)
Biocompatible
Hyperthermia
Can be guided with a magnetic field to desired target
NO-coupled Iron Oxide NPs
NO: molecule used by immune system
Known to be antibacterial
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T. Theivasanthi and M. Alagar, 2010
Zone of Inhibition Test for Antimicrobial
Activity, also called a Kirby-Bauer Test.
It is used clinically to measure antibiotic
resistance and industrially to test the
ability of solids and textiles to inhibit
microbial growth.
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Suresh et al 2013
Copper as a novel biocide
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Synthesis of copper nanoparticles
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There are three main routes—chemical, physical, and biological—being used
for the synthesis of metallic nanoparticles.
These methods for the production of copper nanoparticles are appropriate
for laboratory-scale synthesis but are not economical for a large-scale or
commercial setup.
The induction plasma system (Tekna) has been successfully used in the
synthesis and preparation of advanced materials such as new ceramics,
nanometric metallic powders, biomaterials powders (by HF-75, Tekna).
Making nanopowders by induction plasma (Tekna)
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ICP stands for “inductively coupled plasma”. Also known as induction
plasma, H.F (high frequency) or R.F. (radio frequency) plasma.
Nanopowders synthesis by ICP: Pure metals
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Copper and Copper Oxide NPs
• Average size of CuO NPs is 36.5 ± 16.6 nm
• Average size of Cu NPs is 84.8 ± 24.6 nm for the
core and 17.1± 5.0 nm for the shell 2014 World Sterilization Congress of the WFHSS 19
Copper and Copper Oxide NPs
Minimal Inhibitory Concentration (MIC) for
◦ Cu, S.aureus: 0.325mg/mL
◦ Cu, E.coli: 0.750mg/mL
◦ CuO, both bacterial strains: 0.325mg/mL
At each MIC
◦ suspension was plated and incubated for 24h to determine if NPs are bacteriostatic or bactericidal
◦ Bacteriostatic for both NPs
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Copper and Copper Oxide NPs
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Copper Microparticles (MPs)
MPs average size is 21.31 ± 7.75 m
Smallest microparticle is 6.205 m
Biggest microparticle is 39.7 m.
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Copper Microparticles (MPs)
MIC for S.aureus of MPs is 3mg/mL
◦ Bacteriostatic
◦ At 3mg/mL, suspension was plated and incubated for 24h growth
MIC for E.coli of MPs is 6mg/mL
◦ Bacteriocidal
◦ At 6mg/mL, suspension was plated and incubated for 24h no growth
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Copper Microparticles
S.aureus E.coli
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Discussion
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Cell wall thicker for Gram-positive (many layers of peptidoglycan)
Size effects
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The decline in the biotoxicity level of the
copper compounds in a series:
ions → nanoparticles → microparticles
was observed in good agreement with the
results of animal studies.
Oxidation & Corona
Because Cu is less chemically stable than Cu(II) and
Cu(I) oxides; thus, a major problem is the usual
occurrence of surface oxidation during its synthesis. A
mixture of metallic Cu, cuprous oxide (Cu2O) or cupric
oxide (CuO) generally accompanies the production of
Cu nanopowders.
Uncontaminated and surfactant-free Metal NPs are
difficult to obtain. The bacteria will «see» corona
covering the copper NPs. In fact, the MEM/EBSS or
RPMI-1640 culture media contains inorganic salts,
amino acids, vitamins and other components wich could
«contaminate» these «ultra-pure» NPs.
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Effects of Surface Chemistry on the Generation of Reactive Oxygen
Species by Copper Nanoparticles (Miao Shi et al. 2012, ACS Nano, Vol. 6, No. 3, 2157–2164)
It is essential to develop approaches to control surface chemistry to
better understand the origins of nanoparticle-induced toxicity, as subtle
differences in surface properties can dramatically change biological
responses.
Shi et al studied the ROS generating capacity of uniform copper
nanoparticles with different capping ligands to better understand the
relationship between nanoparticle physicochemical properties and
their toxicological potential as reflected by ROS generation in an
acellular assay. Three mercaptocarboxylic acids with different carbon
chain lengths were used for surface modification as they have been
shown to provide good colloidal stability for various nanoparticles.
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As-synthesized
copper NPs
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TEM micrographs of these copper NPs after surface modification with 8-mercaptooctanoic acid (MOA), 12-
mercaptododec- anoic acid (MDA), and 16-mercaptohexadecanoic acid (MHA).
The ROS generating capacities of these three copper nanoparticle types were measured.
All particles demonstrated dose-dependent increases in ROS activity.
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The ROS generating capacities of the oxidized copper samples. The oxidized copper NPs had
lower equivalent [H2O2] than the corresponding as-made ones.
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IRON OXIDE NPs & MAGNETOSOMES Rational
* SPIONs 1) Positive amine groups 2) Negative carboxylic groups 3) Bare
* MAGNETOSOMES (Natural nanocrystals)
Contrast agents Attachment of NO
NANOSCALE CHARACTERIZATION
19
Methodology
Time-of- Flight Secondary Ion Mass Spectrometry (TOF-SIMS)
Transmission Electron Microscope (TEM)
Fourier Transform Infrared Spectroscopy (FT-TIR)
Vibrating Sample Magnetometer (VSM)
X-ray Photoelectron Spectroscopy (XPS)
http://www.google.ca/url?sa=i&source=images&cd=&cad=rja&docid=U_kcWogVPXj7zM&tbnid=eNwotIki3VYFHM:&ved=0CAgQjRwwAA&url=http://ncem.lbl.gov/frames/oam.htm&ei=KezBUcfNNtKy4AOLjIGoCw&psig=AFQjCNERz84f079gRfAsKwuznzSlwDKt5w&ust=1371749801927489http://www.google.ca/url?sa=i&rct=j&q=FTIR&source=images&cd=&cad=rja&docid=pfkg8HqawP0-bM&tbnid=IyFivoMffX--_M:&ved=0CAUQjRw&url=http://kuthirummaln.people.cofc.edu/&ei=3ezBUYyVHozj4AORwIC4Dw&bvm=bv.47883778,d.dmg&psig=AFQjCNFEngTv-TngmGGjvGGacbo3xV5K2Q&ust=1371749929527462http://www.google.ca/url?sa=i&rct=j&q=&source=images&cd=&cad=rja&docid=vIhTeLb83_OH_M&tbnid=UjGeJK7v8zSQzM:&ved=0CAUQjRw&url=http://www.utdallas.edu/~amy.walker/Facilities.html&ei=NPLBUc6XF9Wz4AO-joHYDw&bvm=bv.47883778,d.dmg&psig=AFQjCNGYOYcoCljCna7PYwVBYOGmv5l7DQ&ust=1371751344577884
TEM SPIONS &
Magnetosomes
TEM
Sample Shape Size
Bare Spherical 9.1 ± 1.4 nm
Negative Spherical 9.7 ± 1.7 nm
Positive Spherical 10.1 ± 1.3 nm
Magneto-
somes
Hexagonal,
square and
spherical
73.7 ± 14.6 nm
length: 0.4-0.8 µm
Figure 5. TEM images of SPIONs a) bare, b) positive, c) negative and d) magnetosomes
Table 2. Shape and Size of SPIONs and magnetosones found by TEM characterization
SIZE DISTRIBUTION
24
Figure 6. Size distribution histograms of SPIONs NPs a) bare, b) positive, c) negative and d) magnetosomes
VMS
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Magnetization
VMS
Sample Mmax
(emu/g) Behavior
Histeresi
s
Positive 28 Superparamag-
netic 0
Negative 24 Superparamag-
netic 0
Bare 27 Superparamag-
netic 0
Magneto-
somes 14.7 Ferrimagnetic 472 Oe
Figure 7. Magnetization curves of SPIONs and magnetosomes
Table 3. Magnetic properties od SPIONs and magnetosones found by VMS characterization
SURVEY MAGNETOSOMES (XPS)
29
XPS
Fe peak ~ 700 eV
Fig 8. Magnetosome survey spectrum. The inset shows the almost complete absense of Fe2p at 710eV
Cytotoxicity : Effect of nanoparticle time of exposition
and dose on cell morphology
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Effect of nanoparticle functionalization on cell viability
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Effect of culture medium on cell viability
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Iron Oxide Nanoparticles
S.aureus E.coli
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Iron Oxide Nanoparticles
E.coli:
◦ No change is notable between control and different concentrations of SPIONs at all time (T1, T3, T24). No effect on
E.coli growth
S.aureus
◦ After 1h, all concentration, decrease bacteria viability is insignificant
• After 3 h, for all concentration, bacteria have a decrement of their
growth arount one log
• After 24h, at smallest concentration (2.5, 1.25) not a real change
between T3 and T24
• 5mg/mL appromatively a decrease of 2log
• 10mg/mL, decrease of 3log
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Nitric oxide
Nitrogen oxide can refer to a binary compound of oxygen and
nitrogen, a mixture of such compounds:
Nitric oxide, also known as nitrogen monoxide, (NO),
nitrogen(II) oxide
Nitrogen dioxide (NO2), nitrogen(IV) oxide
Nitrous oxide (N2O), nitrogen(-I,III) oxide
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IMPORTANCE OF USE NITRIC OXIDE Rational
NITRIC OXIDE
Endogenous
molecule
Vascular tone
Wound healing
Immune
response
Host defense
against infection
NO-releasing
Biomaterials
Inhibiting in vivo
infection
Facilitating tissue
integration of the
implants
Reducing bacterial and
platelet adhesion in vitro
Antimicrobial
properties
G+ G-
Biofilms
P.
aeruginosa
S. aureus S.
epidermidis
E. coli
Klebsiella St.
pneumoniae
DESIGN OF MNPs COATING WITH NO
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Functionalization
Figure 3. Schematic representation of the formation of thiolated Fe3O4 NPs coated with MSA and DMSA
mercaptosuccinic acid (MSA)
dimercaptosuccinic acid (DMSA).
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DESIGN OF MNPs COATING WITH NO
21
NO delivery
Positive SPIONs Negative SPIONs
Figure 4. Schematic representation of iron oxide NPs functionalized with silane layers containing amine groups (positive SPIONs) and carboxilic acid groups (negative SPIONs)
Schematic of two different methods to synthesize diazeniumdiolate-
modified silica nanoparticles. Tetraethoxysilane (TEOS) and N-(6-
aminohexyl)aminopropyltrimethoxysilane (AHAP3) are representative
examples of tetraalkoxy- and aminoalkoxysilanes, respectively. (A)
Postformation method. (B) Pre-formation method (reproduced from ref.
43 with permission from the American Chemical Society).
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Zhang & Yahia
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Time-kill curves for the Staphylococcus aureus arrows, application of a second dose at the same concentration
Plate counting of A. baumannii cells on the wounded skin of mice after 24 h application of SG-80A based NO releasing and control patches. NO releasing and control patches were applied to wounds 24 h after inoculation with A. baumannii. After 24 h, skin tissue was harvested, homogenized, serially diluted and grown on agar plates. The data are means ± SEM (n = 3). ⁄p <
0.05.
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Fredman et al
Schoenfisch et al. Acta Biomaterialia 10 (2014) 3442–3448
NO SH NO /SH
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Concluding Remarks
Various studies have revealed that copper NPs can be
synthesized by chemical, physical, and biological routes.
The chemical methods are time-consuming and tedious.
Moreover, some chemical methods include use of
hazardous chemicals, which may exert adverse effects to
the user.
The induction plasma used for nanopowder synthesis has
many advantages over alternative techniques such as high
purity, ease of scale-up and ease of operation and control.
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Moreover, it was shown that the toxicity of nanosized oxides
(nano CuO) was much high than their bulk counterparts.
Therefore, development of an efficient approach for
preventing the oxidation of Cu nanopowders during synthesis
in an aqueous solution is required urgently.
Inaccuracies of NO measurement methods in biological
media (amperometer NO sensor, chemiluminescence
analyzer --- Significant variations between techniques).
Further investigation on the stability of the stability of the
NO-donors became a significant problem in the particle-
based systems because of the possibility of the release of NO
before reaching the target site.
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Due to the growing tolerance of bacteria to antibiotics, metal and metal oxide nanoparticles with antibacterial properties
represent a promising alternative approach to antibiotics. But
Al2O3 NPs was found to induce bacterial resistance !!!)
Nitric oxide-releasing nanoparticles (NO NPs) and metal-
containing nanoparticles all use multiple mechanisms simul-
taneously to combat microbes, thereby making development of
resistance to these nanoparticles unlikely.
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62 Dr. Yahye Merhi
Dr. Karim Maghni Dr. Theodre Veres
http://www.icm-mhi.org/fr/index.html
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Questions
Félix d'Hérelle
• Mais, en 1917, on le retrouve à l’Institut Pasteur, à Paris, où il fait une
découverte majeure : le bactériophage, un ultravirus (impossible à observer
à l’époque, car précédant la mise au point des microscopes électroniques)
qui s’attaque aux bactéries. Il en conclut à la possibilité d’utiliser ce
« microbe invisible » pour combattre toutes les épidémies. Sur la base de
l'article publié dans The Lancet par Frederick Twort en 1915, d'Hérelle
publie, en 1917 son fameux article princeps : Sur un microbe invisible
antagoniste des bacilles dysentériques (Comptes rendus de l'Académie des
Sciences, Paris, 1917. 165 :p. 373-5.
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http://fr.wikipedia.org/wiki/Institut_Pasteurhttp://fr.wikipedia.org/wiki/Institut_Pasteurhttp://fr.wikipedia.org/wiki/Institut_Pasteurhttp://fr.wikipedia.org/wiki/Parishttp://fr.wikipedia.org/wiki/Bact%C3%A9riophagehttp://fr.wikipedia.org/wiki/Microscope_%C3%A9lectroniquehttp://fr.wikipedia.org/wiki/Microscope_%C3%A9lectroniquehttp://fr.wikipedia.org/wiki/Microscope_%C3%A9lectroniquehttp://fr.wikipedia.org/wiki/The_Lancethttp://fr.wikipedia.org/wiki/The_Lancethttp://fr.wikipedia.org/wiki/The_Lancethttp://fr.wikipedia.org/wiki/Frederick_Tworthttp://fr.wikipedia.org/wiki/Frederick_Tworthttp://fr.wikipedia.org/wiki/Frederick_Twort
• La paternité de la découverte des
bactériophages est souvent disputée entre
Frederick Twort et Félix d'Hérelle. La
publication de Twort dans The Lancet en 1915
est toutefois très différente de celle de Félix
d'Hérelle en 1917.
• Une collection Félix-d’Hérelle comprenant
420 virus a été montée par Hans Wolfgang
Ackermann, professeur à l’université Laval.
En 2003, au moment de sa retraite, cette
collection a été transférée à Sylvain Moineau,
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http://fr.wikipedia.org/wiki/Bact%C3%A9riophagehttp://fr.wikipedia.org/wiki/Frederick_Tworthttp://fr.wikipedia.org/wiki/The_Lancethttp://fr.wikipedia.org/wiki/Universit%C3%A9_Laval
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Tawil et al
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Tawil et al
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Tawil et al
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Schematic illustration of various possible orientations of phage on surface. The bacterial capture
proteins are marked in green (a) tailed phage can adsorb head-down,tail-down or side-ways, (b)
icosahedral asymmetric phage, (c) filamentous phage can adsorb via either pole or side-ways, (d)
filamentous phage are prone to bundling oraggregation (left), efforts to orient them typically focus
on arranging them parallel on the substrate (right).
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Various methods of orienting phage: (a) electrostatic, (b) assembly at liquid–liquid interface,
reproduced with permission from Ref. [163], (c) molecular imprinting Ref. [167] (d) molecular
imprinting from Ref. [166]
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(a) SEM pictures of sample 3 copper nanoparticles;
(b) TEM pictures of sample 3 copper nanoparticles.
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Effects of Surface Chemistry on the Generation of Reactive Oxygen
Species by Copper Nanoparticles (Miao Shi et al. 2012, ACS Nano, Vol. 6, No. 3, 2157–2164)
It is essential to develop approaches to control surface chemistry to better
understand the origins of nanoparticle-induced toxicity, as subtle differences in
surface properties can dramatically change biological responses.
Shi et al studied the ROS generating capacity of uniform copper nanoparticles
with different capping ligands to better understand the relationship between
nanoparticle physicochemical properties and their toxicological potential as
reflected by ROS generation in an acellular assay. Three mercaptocarboxylic
acids with different carbon chain lengths were used for surface modification as
they have been shown to provide good colloidal stability for various
nanoparticles.
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As-synthesized
copper NPs
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TEM micrographs of these copper NPs after surface modification with 8-mercaptooctanoic acid (MOA), 12-
mercaptododec- anoic acid (MDA), and 16-mercaptohexadecanoic acid (MHA).
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The ROS generating capacities of these three copper nanoparticle types were
measured. All particles demonstrated dose-dependent increases in ROS
activity.
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The ROS generating capacities of the oxidized copper samples. The oxidized copper
NPs had lower equivalent [H2O2] than the corresponding as-made ones.
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Other applications
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Schematic of the atherosclerosis treatment with LDL and ox-LDL removal from the bloodstream
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Iron carbides. The carbon surface can be chemically functionalized using physisorbed poly(ethylene imine) and iminodiacetic acid (d) or thiol-moieties which can be crosslinked with antibody fragments using a poly(ethylene glycol)-based crosslinker (e).
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