In vitro study of nanobiomaterialsbiocompatibility

Marie-Françoise HARMAND

LEMI – Technopole Bordeaux Montesquieu – 33650 Martillac – France

DETERCA – Université Victor Segalen Bordeaux 2 – 33076 Bordeaux Cedex - France

Nanomaterial :Particles with lengths that range from 1 to 100 nanometers in twoor three dimensions.ASTM E 2456-06 : Terminology for nanotechnology. ASTM International 2006.

Biomaterial :A non-living material intended to interface with biological systems to evaluate, treat, augment or replace any tissue, organ or function of the body.Chester 1986.

Nanobiomaterial :A biomaterial substrate composed of nanometer-scalecomponents.Example : the inorganic bone matrix is comprised of apatite crystals withfinal dimensions 400 x 30 x 75 Å.

Interest

• Cells in our body are predisposed to interact with nanostructuredsurfaces developing subtle biomimetism.

• Proliferation, migration and extracellular matrix production by cells during tissue repair are dependent on protein adsorption on the surface of implanted biomaterials.

• Protein adsorption characteristics are in turn dependent on the surface features of implanted biomaterials. Integrins are transmembrane cell receptors interacting with the protein layer adsorbed to the scaffold. These interactions are governed by molecular events at nanometer scale.

• Nanobiomaterials have an increased number of atoms at their surface and possess a higher surface area to volume ratio than conventional microscale biomaterials.

• Thus scientific developments of nanobiomaterials are multiple, from bone implants to bone (or cartilage) tissue engineering scaffolds, to drug release.

Biocompatibility

Biocompatibility was defined at the ESB Concensus conference (Chester – 1986) as « the ability of a material to performwith an appropriate host response in a specific application ».

The development of a risk management system for biomaterials was developped in the framework of two international standards :

– ISO 14971 « Medical devices – Riskmanagement ».

– ISO 10993 « Biological evaluation of MedicalDevices » which specifies requirements and gives guidance on procedures to be followed in the evaluation of the potential for medical (or dental) materials to cause adverse health effect.

In ISO 10993 the specificity of nano-biomaterials is not taken into account.

• The purpose of this talk is to try to present a base set of biocompatibility tests for nanobiomaterials, adapted from the existing risk systems of reference.

• The potential risk of nanobiomaterials is exposure of human tissues to migrating nanoscale particles :– in the work place during the fabrication

(pulmonary, dermal, oral or ocular routes) ;– after implantation of nano-particles (drug

release, dendrimers) or of biomaterials consisting of nanoparticles, through their soluble or insoluble degradation products.

Borm et al. Toxicol Sci. 2006; 90(1):23-32

Risk analysisBiocompatibility study

1st step

Nanobiomaterial physicochemical characterization

– Size and size distribution– Crystal structure– Chemical composition– Surface energy

– In vitro biodegradation or dissolution (part of ISO 10993)– In vitro protein adsorption study

Risk analysisBiocompatibility study

2nd step« In vitro » biocompatibility assessment

– Cytotoxicity : cell viability, apoptosis vs necrosis – ISO 10993-5

– Genotoxicity – ISO 10993-3

• Ames test (Bacterial reverses mutation) – OECD 471• Chromosome aberrations in human lymphocytes – OECD 473• MLA – OECD 476

– Carcinogenicity using cell transformation assay.

– Hemocompatibility - ISO 10993-4 :

• Hemolysis, PTT, Platelet activation

• Complement acivation

– Phagocytosis using primary human cell cultures

– Monocyte – macrophage activation (cytokine release)

Risk analysisBiocompatibility study

3rd step

« In vivo » biocompatibility assessment

– Acute oral toxicity (OECD 423)– Acute inhalation toxicity (OECD 403) (biochemistry,

hematology, histopathology of the major organs)– Acute dermal irritation/corrosion (OECD 404)– Acute eye irritation/corrosion (OECD 405)– Skin sensitization (OECD 406)

Risk analysisEcological effect

4th step

Aquatic screening battery

– Rainbow trout (OECD 203)– Daphnia (OECD 202)– Green algae (OECD 201)

Cytotoxicity of single-wall carbonnanotubes on human fibroblasts

Tian F, Cui D, Schwarz H, Estrada GG, Kobayashi H.

Toxicol In Vitro. 2006 Oct;20(7):1202-12.

(2 nm x 500 nm)

A novel method to prepare water-dispersible magnetic nanoparticles and their biomedical applications: Magnetic

capture probe and specific cellular uptake

Yu C, Zhao J, Guo Y, Lu C, Ma X, Gu Z

J Biomed Mater Res A. Published on line 7 Jan 2008

• Water-dispersible MNPs were prepared by modification of the oleic acid capping groups on the surface of iron oxide nanoparticleswith glyco-saminic acid (GA) to give GA-MNPs.

• The cellular uptake of the GA-MNPs into mouse macrophage cells, mouse embryonic fibroblast cells and two kinds of human cancer cells was carried out. The results demonstrate that the GA-MNPs have great potential to be used as contrast agents for cancer diagnosis.

In vitro cytotoxicity, hemolysis assay, and biodegradationbehavior of biodegradable poly(3-hydroxybutyrate)-

poly(ethylene glycol)-poly(3-hydroxybutyrate) nanoparticlesas potential drug carriers.

Chen C, Cheng YC, Yu CH, Chan SW, Cheung MK, Yu PH.

J Biomed Mater Res A. Published on line 7 Jan 2008

Cisplatin-loaded Au-Au(2)S nanoparticles for potential cancer therapy: Cytotoxicity, in vitro

carcinogenicity, and cellular uptake

Ren L, Huang XL, Zhang B, Sun LP, Zhang QQ, Tan MC, Chow GM

J Biomed Mater Res A. Published on line 26 Sep 2007

Cyto- and genotoxicity of ultrafine TiO2particles in cultured human

lymphoblastoid cells

Wang JJ, Sanderson BJ, Wang H.

Mutat Res. 2007 Apr 2;628(2):99-106.

In conclusion, the present study shows thatUF-TiO2 can induce significant cytotoxicityand genototxicity in cultured human cells. However, the precise mechanism of MN, apoptosis formation and inhibition of celldivision by UF-TiO2 is unclear. Additionalwork needs to be undertaken to understandthe mechanism of damage.

Tsuji et al. Toxicol Sci. 2006; 89(1):42-50

Toxicol Sci. 2006;89(1):42-50

• The application of nanotechnology to biomaterial science is a great potential, however the risk to human health and environment must not be overlooked : safe fabrication, commercial scale processing, use as implants.

• Continuous monitoring is necessary to assess the potential risk of newly designed nanobiomaterials.