dr. sc. ivana vinković vrček · 2019-04-09 · 2) advantages vs. disadvantages? 3) what should be...

Dr. sc. Ivana Vinković VrčekInstitute for Medical Research and Occupational Health Zagreb, Croatia

2nd largest Croatian public research institute

Research fields: ◦ Biomedicine◦ Toxicology◦ Occupational health ◦ Environmental safety

Professional services: ◦ analytical, monitoring, toxicological, regulatory consulting

2

Niche: quality/efficacy/safety evaluation of theranostics

Innovative R&D topics: ◦ Personalised protein corona – innovative way for nanodiagnostics

◦ Biocidal nanmaterials (Ag, Se, Au….)

◦ Nanoparticles in hypoxia (focus on neurodegenerative diseases)

◦ Blood-brain barrier (in vitro, in vivo; high throughput)

Technical expertise: ◦ Synthesis and characterization of nanoparticles (Au, Ag, SPIONs, Se, TiO2)

◦ Molecular biology and cell culture laboratories, in vitro/in vivo testings;

◦ Electron and fluorescence spectroscopy, mass spectrometry, nuclearmagnetic resonance, MALDI-TOF imaging

◦ Computational methods (molecular dynamics, quantum mechanics, QSAR)

3

Design of different

metallic NPs

Physico-chemical

transformations

Abiotic transformations

Biotic transformations

In vitro toxicity

evaluation

In vivo toxicity

evaluation

NANOSAFETY

Same properties of nanoparticles that are desirable and potentially useful from a technological or biomedical perspective are also the properties that may give rise to hazardous, unexpected toxicities

What is Safe-by-Design approach?

What are regulatory requirements for clinicaltranslation of nanomaterials: regulation on medicines vs. regulation on medical devices?

What analytical techniques and methodology are on disposal for quality, efficacy and safety (QES) evaluation of nano-enabled medical products?

What are their advantages vs. disadvantages?

What should be defined by QES evaluation to implement Safe-by-Design approach?

Can Safe-by-Design approach really foster clinicaltranslation of nano-enabled medical products?

1st Action Training School, Trieste April 8-11, 2019

What is Safe-by-Design approach?


Developed in NanoReg2

The three pillars underpinning Safe by Design: Safe design, Safe production, and Safe use

„The 'Safe-by-Design' concept aims at reducing potential health and environmental risks at an early phase of the innovation process. The concept aims at creating an integrated research strategy, which enables the consideration of safety aspects for humans and the environment in the design process of a product/material to eliminate or minimise the risk of adverse effects during its life cycle including construction, use, maintenance and deconstruction. „


Closely linked to the Stage Gate model.

8

◦ to reduce uncertainties associated with nanomaterials while they are still in development - before they reach any market application

◦ to ensure that

the risks of products launched in the market are known and managed,

the predicted benefits outweigh any residual risks,

industrial actors reach a situation of regulatory preparedness as their products develop

◦ to enhance the public trust that innovators care about human health and environmental safety in addition to their profit margins


• benefits◦ early and easier identification of uncertainties and risks◦ reduction of uncertainties and risks◦ preparedness to meet todays and future regulatory

requirements◦ well-balanced safety, functionality and costs◦ better design of products and better business models

provides a safety net…◦ for innovators to avoid confrontation with safety/regulatory

issueslater on in the innovation process,

◦ for investors and insurers to minimize uncertainty about health risks,

◦ for regulators to minimize casualties,◦ for society to benefit from safer innovative products.


aims at reducing potential health and environmental risks at an early phase of the innovation process →’Design Phase’

’Design Out’ – hazardous properties to minimize

possible risks from the very beginning

Integrating safety consideration into design phases

Product

idea

Research &

designPrototype Testing

Manufactu-

ring

Design safeand functional

materials

Safetyassessment


What are regulatory requirements for clinical translation of nanomaterials:

regulation on medicines vs. regulation on medical devices?


Safety Assessment according to REACH◦ Collection and evaluation of all available and

relevant information on the used substance in order to identify potential hazards of the substance

◦ Safety data sheets

◦ EC recommendations

◦ Exposure limit values

◦ Peer reviewed data, scientific literature, relevant databases

◦ Lack of data further tests needed


EMA working definition of Nanomedicines:

◦ Purposely designed systems for clinical applications

◦ At least one component at nano-scale size

◦ Resulting in definable specific properties and characteristics related to the specific nanotechnology application and characteristics for the intended use (route of admin, dose)

◦ associated with the expected clinical advantages of the nano-engineering (e.g. preferential organ/tissue distribution)

And needs to meet definition as a medicinal product according to European legislation.


Address unmet medical needs Integrate efficacious molecules that otherwise

could not be used because of their high toxicity (e.g. Mepact)

Exploit multiple mechanisms of actions (e.g. Nanomag, multifunctional gels, polymers in development)

Maximise efficacy and reduce dose and toxicity Drug targeting Controlled and site specific release Preferential distribution within the body (e.g. in

areas with cancer lesions) Improved transport across biological barriers


Source:

http://science.nasa.gov/headlines/y2002/15jan_nano.htm

Source: http://foresight.org/Nanomedicine/Gallery/index.html

Artery cleaners

Nanoprobes for viruses

Nanobots to replace damaged neurons

Source: http://www.e-spaces.com/Portfolio/trans/blood/index.html

Nanoparticles in medicine

theranostic systems for more personalized and precision healthcare

http://www.bvis.uic.edu/bvis/gallery/student/modeling02.htm

http://www.bvis.uic.edu/bvis/gallery/student/modeling02.htm

As for any medicinal product, the EU competent authorities will evaluate any application to place a nanomedicinal product on the market, utilisingestablished principles of benefit/risk analysis, rather than solely on the basis of the technology per se” (including RMP and enviromental risk assessment) Reflection paper on nanotechnology-based medicinal products for human use


In April 2017, the European Parliament and Council adopted a number of

important legislation changes that will improve the safety and efficiency of

medical devices (MDR 2017/745) and in-vitro diagnostics (IVDR 107/746).

These regulations entered into force on the 25th of May 2017.

Legislation changes will become effective by 2020, giving companies limited time to become fully compliant


The legislation changes will ultimately:◦ Provide greater surveillance and management of the

entire life cycle of Medical Devices and IVD’s◦ Increase clinical investigation to ensure patient

safety as priority◦ Promote better post-marketing surveillance◦ Encourage transparency and traceability between

economic operators◦ Increase requirements for technical documentation◦ Expand and clarify classifications and definitions to

reduce ambiguity◦ Manage risk to ensure overall patient safety


Cosmetic contact lenses without intended medical purpose Equipment used for liposuction/lipolysis or lipoplasty Epilation equipment and hair removal lasers Conception control/support (condoms and intrauterine devices) Any software used for anything defined as a medical device Products used for cleaning, sterilisation and disinfection of medical

devices Accessories that enable a medical device to be used for intended purpose

Apart from medical device manufacturers, other stakeholders will be affected by the new legislation. The role of regulatory agencies such as the Notified Bodies and Competent Authorities will now implement more rigorous control in order to enforce regulatory compliance. In particular Notified Bodies will become more active operators, carrying out more inspections, sample checks and audits to encompass the entire life cycle of the device.Due to increased transparency and the creation of accessible databanks,patients will have access to additional information regarding the safety andinstructions for intended use of medical devices.


Legacy products must be reviewed and some may need reclassification

Approximately 90% of IVDs must be reviewed (increased from just 10% in 2017)

A unique Device Identifier (UDI) must be implemented for every device

Increased role of regulatory bodies – unannounced audits!

Increased clinical data requirements Increased requirements for post-marketing

surveillance Increased liability for manufacturers (greater quality

assurance)


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Scientific Advice and Protocol Assistance


Exposure characterisation: Differences between properties of as-produced /suppliedmaterials vs. administered materials vs. materialsfollowing administration

Nature of NM may change during the life cycle Lack of sufficient knowledge of how NM behave in the

body and in the environment:◦ In real matrices (biological tissues, food, consumer products

many methods not applicable or meaningless (agglomeration, binding of NP to matrix, presence of bio(geo)genic NP

Each particle may behave differently Political/regulatory constraints on the use of in vivo

testing How to define dose? Mass per volume, Number per volume,

Moles per volume

More questions than answers!251st Action Training School,

Trieste April 8-11, 2019

Quality, efficacy and safety (QES) evaluation of nano-enabled medical products :

Characterization! Stability, biotransformation and nano-bio interactions! In vitro testings! In vivo testings!

1) Analytical techniques and methodology on disposal?2) Advantages vs. disadvantages?3) What should be defined by QES evaluation to implement Safe-by-Design approach?4) Can Safe-by-Design approach really foster clinicaltranslation of nano-enabled medical products?


The tailorability of nanoparticles

UNDERSTANDING BIOLOGICAL RESPONSE

Characteristics

• Size and shape

• State of dispersion

• Chamical composition, crystallinity, solubility, impurities

• Surface area andporosity

• Surface properties

Consequences

• Thermodynamicstability

• Aggregation/agglomeration behaviour

• Interaction withsurrounding matrix

• Ageing

• Adsorption of ions

• Catalytic effects

Effects in livingsystem

• Translocation

• Interaction withbiomolecules

• Formation of reactiveoxygene species

• Protein binding

• Accumulation andretention

• Cell/tissue response

AgNPs AgNPs in cellculture media

AgNPs in cellculture media+ albumin

28

LIST OF PHYSICO-CHEMICAL PARAMETERS

Physico-chemical parameters Possible techniquesChemical composition/Identity A wide range of analytical methods, including

UV -Vis, HPLC, GC/LC -MS, AAS, ICP-MS, FTIR, NMR, XRD, etc

Particle size (primary/secondary and size distribution)Structure: aggregation and agglomerationParticle and mass concentration

FFF, HDC, HPLC, AUC, CPS disc centrifugation, TEM, SEM, AFM, DLS, DMA

Morphology: shape, surface area, surface topology (roughness), crystallinity, porosity

AFM, TEM, SEM, NMR, XRD, BET

Surface chemistry: zeta potential/surface charge, surfacecoating, functionalization, catalytic activity

LDE, SPM, XPS, MS, RS, FTIR, NMR, AUC (for surface composition), GE, SPM, LDE, PALS (for zeta potential), Nano SIMS, SERS

Redox potential, Solubility, Dustiness, Density, Viscosity Potentiometric methods, X-ray absorption spectroscopy

Stability MS, HPLC, DLS, FTIR, NMR

Photocatalitic activity, UV absorption (extinctioncoefficient), light reflection

Spectroscopic methods (UV-Vis, fluorescence…)

IMAGING TECHNIQUES

• Possibility to see target analyte

• Determination of size and shape

• Coupling to EDX/EDS elementalcomposition

• Various techniques

Advantages Drawbacks

Scanning Electron Microscopy(SEM) / Transmission ElectronMicroscopy (TEM)

Accesible size < 1 nm, very highresolution; direct method; no calibration necessary; anyparticle shape is accessible

Expensive and complexequipment; high vacuum is needed; sample preparation; time-consuming, poor statistics; artefacts; matrix effects

Atomic Force Microscopy (AFM) Fast; equipment readilyavailable; inexpensie; very highresolution

Statistics problems; stronginfluence of the tip (size, shape, material); influence of the NP type (by tip-particle interaction); partiles have to be on a surface

30

Light scattering

• Widely used

• Various techniques (static(SLS), multi angle laser (MALLS), dynamic (DLS)

• Used as detector aftersize separation (e.g. FFF)

Advantages Drawbacks

Inexpensive, fast, goodstatistics,no influence ofbeam, possibleweighting for intensity, volume and numberpossible

Indirect measurement,only dispersed samplescan be measured, influence of medium, “spherical”particles, no model for veryelongated or irregularshapes

1 10 100 1000

mean

vo

lum

e %

d / nm

Domazet Jurašin et al., Beilstein J Nanotechnol, 2016, 7, 246.

31

FORMATION OF PROTEIN CORONA


citrate-coated AgNPs

PVP-coated AgNPs

32

Up to now: detailed analysis only of „hard corona”

Procedure:

Incubation of NP with protein-rich

mediaCentrifugation Washing

Elution of bound

proteins

Analysis(SDS-

PAGE, LC-MS)

Several times

Challenge!

Nonperturbing methods that do not

o disrupt protein-NP complex,

o change kinetic and thermodynamic control,

o induce additional protein binding.

Problems?

o The concentration of the particle probes and proteins is artificially increased during centrifugation

o Does corona change during analysis?

o Significant non-specific interactions caused by the separation process

centr

ifuge


(a) Cellular level (cytotoxicity) – apoptosis, necrosis, growth arrest, abnormal morphology, undesired cell signaling or secretory activity. Thorough understanding of these mechanisms and events requires analyses at even more discrete levels:

Molecular level – interaction of UCNPs with proteins, cell signaling or mitochondrial electron transport, mutational alterations, reactive oxygen species (ROS), DNA damage, mRNA degradation, gene expression, etc.

Subcellular level – membrane disruption or permeability changes,mitochondrial activity, apoptosis.

(b) Organ level –effects on different organs (kidney, spleen, liver, heart, brain, lungs, skin) assessed or observed after a certain period of exposure.

(c) Whole organism – assessment of the overall body condition, symptoms of abnormal behavior, changes in reproductive potential etc.

(d) Environmental – perhaps the most difficult area of nanotoxicology research due to its complexity.


Assessment ofcytotoxicity over a

range ofconcentration

Investigation ofmechanism

underlying toxicity

Assessment of realibilityand reproducibility of the

protocols


Source of variability Description

Cell maintanance includes variability in the maintenance of a cell line such as the following: cell passage number, cell freeze passage, passaging procedure, cell vendor, serum vendor and lot number differentDNA/genotype

Pipetting Addresses differences in pipetting reproducibility from one well to the other due to the pipetting process. Includes differences in cell seeding density, reagent volume (either of the disturbant of interest or finally the MTS assay reagent)

Instrument performance

Addresses issues concerning nonlinearity or general functional problems with the instrument needed for assay readout.

Toxic chemicalpositive control

represents the sources of variability in a toxic response to a positive control reference material. Many of these sources are common for the chemical control and ENM testing system. This branch serves as an assay test system performance control.

Assay protocol includes conditions and protocol specifications, which can influence the mechanistic part of the assay readout such as the following: age, storage temperature, and freeze/thaw-cycle numbers of the assay reagent change in background absorbance optical degradation of reagents

UCNPs handling andcharacterisation

includes all aspects of ENM: dispersion method and quality, physicochemical properties (e.g., surface charge and chemistry, surface area and reactivity, size, shape, etc.), agglomeration behavior in cell-culture medium interference reactions with the assay itself (e.g., quenching events)

Roesllein et al., Chem Res Toxicol, 2015, 28, 21.

Assessment ofcytotoxicity over a

range ofconcentration

Investigation ofmechanism

underlying toxicity

Kong et al. Chem. Res. Toxicol. 2015, 28, 290-295:„Standard toxicity assays, initially developed for the evaluation of direct interaction between chemicals and cells, are often inadequate for nanotoxicity assessment. In nanotoxicology, we should consider the indirect interaction betweennanomaterial-component complexes and cells.”


Cell viability Growth activity (Proliferation assay)

Plating efficiency (colony forming ability)

DNA synthesis Thymidine incorporation

Metabolic activity/pathway methods Western blot, Trypan Blue, PI uptake (flow cytometry), MTT (spectrophotometry), Neutral Red Uptake, WST-1 assay, Alamar Blue test

Cell death Apoptosis: Annexin V-FITC/PI Staining, Loss of mitochondrial membrane potential by flow cytometry Necrosis: LDH


Methods:

ROS detection with plate reader or flow cytometry,

Reagents: DTT, Thiol detection, Hemoxygenase

(qPCR), DCFH-DA Free radical formation,

Dihydroetidium (DHE) oxidation, Bromobimane

Nitric oxide assay, Catalase, Glutathione S-

transferase, Glutathione peroxidase


Methods:

Comet Assay +

Comet Assay with

detection of

oxidative purines

(FPG)

Cytokinesis Block

Micronucleus

Assay (CBMN)


In cell free system – readouts

observed after incubation of

tetrazolium reagent with NP

dispersions

0

200

400

600

800

1000

1200contr

ol

0,1 1 5

10

50

10

0

0,1 1 5

10

50

10

0

0,1 1 5

10

50

10

0

0,1 1 5

10

50

10

0

0,1 1 5

10

50

10

0

0,1 1 5

10

50

10

0

0,1 1 5

10

50

10

0

mg/L citAgNP mg/L aotAgNP mg/L pvpAgNP mg/L brijAgNP mg/L tweenAgNP mg/L pll/AgNP mg/L ctabAgNP

% o

f contr

ol fo

rmazan

MTS MTT

0

50

100

150

200

250

300

350

400

co

ntr

ol

0,1 1 5

10

50

100

0,1 1 5

10

50

100

0,1 1 5

10

50

100

mg/L unIONP mg/L manIONP mg/L pllIONP

% o

f co

ntr

ol fo

rma

za

n

MTT CCK


In cell free system – readouts observed

after incubation of tetrazolium reagent

with NPs and subsequent centrifugation.

0

20

40

60

80

100

120

co

ntr

ol 1 5

10

50 1 5

10

50 1 5

10

50 1 5

10

50 1 5

10

50 1 5

10

50 1 5

10

50

mg/L citAgNP mg/L aotAgNP mg/L pvpAgNP mg/L brijAgNP mg/LtweenAgNP

mg/L ctabAgNP mg/L pllAgNP

% c

on

tro

l fo

rma

za

n

MTT CCK

0

20

40

60

80

100

1 5 10 50 1 5 10 50 1 5 10 50

mg/L unIONP mg/L manIONP mg/L pllIONP

% c

on

tro

l fo

rma

za

n


The incubation of Trolox with DCFH is known to result in a time-

and dose-dependent increase in DCF fluorescence. During

incubation, the abstraction of hydrogen from DCFH to the Trolox

phenoxyl radical resulted in the formation of fluorescent DCF.

Upon addition of AgNPs or g-Fe2O3NPs in incubation medium

of DCFH and Trolox, DCF fluorescence quenching was

observed only for neutral and negatively charged AgNPs,

while g-Fe2O3NPs and positively charged AgNPs caused an

enhancement of the fluorescence signal

In cell free system - NPs were incubated with Trolox and DCFH, and fluorescence was

measured after 10, 20, 30, 40 min, 4 and 24 h.


Cytotoxicity assay Detection

principle

Altered readout NP

interference

MTT – cell viability Colorimetric detection

of mitochondrial

activity

Reduced indication

of cell viability

Adsorption of

substrate or dyeMTS – cell viability

CCK-8 – cell viability

DCFH – stress

response

Fluorimetric detection

of ROS production

Reduced indication

of oxidative stress

Fluorescence

quenching

DHE – stress

response

MBCl – stress

response

Fluorimetric detection

of intracellular GSH

Reduced or no

indication of GSH

level changes

Adsorption of

GSH

Source: Vinković Vrček et al. RSC Adv. 2015, 5, 70787.


Finding right dose for in vivo NP exposure

•The dose should mimic the actual quantity of NP exposure to humans

•Determination of the actual dose - problematic owing to their small size and quantity

•The dose of NP for in vivo exposure should not exceed a limit that enhances agglomeration

Finding appropriate vehicle and optimization

of NP dispersion

•NPs are prone to agglomeration at physiological salt concentration and pH due to their increased relative surface area

•Selection of an appropriate vehicle and standardizationprotocol for dispersion conditions (may vary depending on the route of exposure and the source of NPs)

Interaction with biostructures after in

vivo dose delivery

•Changes in cellular components, salt and pH will have an impact on the agglomeration status of NP

•Covering the NPs with proteins may affect their biodistribution and subsequent interaction with cells/tissues

•May trigger conformational changes in protein folding, altering their biological functions; may affect signalling pathways activated by NPs


Routes of administration

OralAdvantages: non-invasive

Disadvantages: 1st passthrough gastric and hepatic

system, potentialtranslocation into systemiccirculation, requires intact

intestinal mucosa

TransdermalAdvantages: non-

invasive, large surface area, local action

Disadvantages: localirritation, potential

translocation into systemiccirculation

InhalatoryAdvantages: non-

invasive, large surface area, local action, avoidance of

metabolic system

Disadvantages: localtoxicity, potential

translocation into systemiccirculation

IntravenousAdvantages: systemic

delivery, systemic action

Disadvantages: 1st passthrough gastric and hepatic

system, systemic toxicity


Dermaladministration

Inhalatoryadministration

Oraladministration

Skin Respiratory tract GI-tract

Brain

Other organs

Heart Kidney Liver

Bile

Central bloodcirculation

ABSORPTION

DISTRIBUTION

METABOLISM, EXCRETION Urine Feces

ADME


PVP AgNP

LD = 0.1 mg Ag/kg b.w. HD = 1 mg Ag/kg b.w.

/ 0.5 ml by oral gavage

ORAL, 28 DAYS

28th DAY narketan/xilapan anesthesia

Blood and organs

6 weeks old (b.w. cca 130 – 150 g)

THE ORGAN DISTRIBUTION PATTERN OF SILVER IN FEMALE AND MALE WISTAR RATS

HIGH Ag

LOW Ag

LOW DOSE HIGH DOSE

EPIDIDYMISBRAINLIVERKIDNEYTESTISHEART

HEARTEPIDIDYMISBRAINLIVERKIDNEY TESTIS

LOW DOSE HIGH DOSE

LIVERKIDNEY BRAINOVARIESHEART

KIDNEY LIVERBRAINOVARIESHEART

IN VIVO RESPONSE TO AgNPs !


PVP AgNP

LD = 0.1 mg Ag/kg b.w. HD = 1 mg Ag/kg b.w.

/ 0.5 ml by oral gavage

ORAL, 28 DAYS

28th DAY narketan/xilapan anesthesia

Blood and organs

6 weeks old (b.w. cca 130 – 150 g)

TEM micrograph of liver excised from treated male rat!

IN VIVO RESPONSE TO AgNPs !


AOTAgNPs in liver

AOTAgNPs in kidney

BSAAgNPs in kidney

PLLAgNPs in liver PVPAgNPs in liver

PVPAgNPs in kidney

PLLAgNPs in kidneyFor dispersions of AgNPs in liver homogenates, healthy 12-week-old male Wistar rats were euthanized by narketan/xilapan anesthesia following the tissue collection. Then, different AgNPs were dispersed in 1 mL of 10 % liver homogenate at final metal concentration of 10 mg L-1 and agitated for 30 min on digital waving rotator. After incubation, suspensions were diluted 50 times before further analysis. TEM samples were prepared by depositing a drop of the NPs suspension on a Formvar® coated copper grid and air-drying at room temperature.

Behaviour of sodium bis(2-ethylhexyl)-sulfosuccinate-coated (AOTAgNPs), poly-L-lysin coated (PLL AgNPs) and polyvinylpyrrolydone coated silvernanoparticles (PVPAgNPs) in liver homogenates


Whole blood Blood plasma

AOTAgNPs in WhBl AOTAgNPs in BlPl

BSAAgNPs in WhBl BSAAgNPs in BlPl

PLLAgNPs in WhBl

PVPAgNPs in WhBl PVPAgNPs in BlPl

PLLAgNPs in BlPl

Nano-bio interface


citrate-coated AgNPs

PVP-coated AgNPs


degradation

hydrophilic

or

hydrophobic

interactions

cation

or

anion

binding

competitive

protein

binding

dissolution

accumulation,

agglomeration

nanoparticle

(NP)

surface

reconstructionBio

tran

sfo

rmat

ion

of

NP

sin

vivo


Biotransformation of AgNPs in biological media

Medium dH (nm) % Volume ζ (mV) % Ag+

MQ water 4,99 ± 0,56

31,16 ± 3,54

98,36

1,64

-18,02 ± 3,71 0,74

Cell culture

media

20,7 ± 3,7

68,7 ± 11,7

506,6 ± 141,2

13,3

18,1

68,6

-8,2 ± 0,4 0,85

Lysosomal fluid 7,4 ± 1,2 100,0 -8,6 ± 1,0 1,73

Gastric fluid 9,2 ± 2,1

48,0 ± 2,7

98,9

1,1

0,1 ± 0,9 4,30

Hydrodynamic diameters (dH) and corresponding volume percentages, zeta potential and percentage of released Ag+ for PVP AgNPs after 1h exposure in different media.

Evidence for formation ofAgNPs in the presence of GSH. 1H NMR spectra of GSH andGSH-AgNP in phosphate buffer(pH 7). GSH-AgNP spectrumwas recorded after 24 h ofAgNP synthesis with GSH as a reducing agent.

GSH

GSH-AgNP

3

3

4

4

7

7

9

9


2O

2O

e–

oxidative damage

interaction

with

biointerfaces

protein crowding

and layering

conformational

changes

nanoparticle

(NP)

steric

hindrance

Effe

cts

of

NP

sin

vivo


Does certain protein corona increase biocompatibility of AgNPs?Study on SOD expression in different organs of female Wistar rats after 30 min exposure to AgNPs stabilized with different surface coatings

Synthesis of MT-coated

AgNPs

Blood and

organs

Injection into

abdominal vein

Collection

after 30 min

exposure

Metallothionein(MT) extraction

28th DAY

narketan/ xilapan

anesthesia

Liver &

kidney

12 weeks old (b.w. 200 g)

control citrate-coated

PVP-coated

albumin-coated

MT-coated


Risk/benefitratioassessment

Available andaplicable exisitingframeworksLimited scientificinformationFirst global approaches for screeeningassessment

Exposure uncertainities due to uncertain fateUncertain effects treshholdsUncertainity of riskcharacterization metricsTools for location-specificassessment

Evaluation of product vs. NPs vs. aged NPsAddress physical form and spatialvariabilityInvestigate interactions with toxicchemicalsConsider NPs-type specificmetrics

SbD implementation - remaining challenges

State-of-the-art Gaps Needs

Detection andcharacterization

In prisitineconditions

Complex mediaRealistic concentrationAged NM

Reliable techniquesMetricsReference materials

Predicition ofNPs fate in vitroand in vivo

Behaviour i laboratory settings

Nature of released, alteredand aged NPs

Information and techniques for altered NPs in complex media

Hazardassessment

Endpoints andrelevant species

Sufficiently fast and targetedanalytical methodologyAppropriate controlsTime-dependent and long-term exposureScale (volume) problems

Technology for exposuremonitoringTime-varying exposurePrioritization of toxicology testsDevelopment of minimum toxicology recommendations

dr. sc. ivana vinković vrček · 2019-04-09 · 2) advantages vs. disadvantages? 3) what should be...

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