A comparison of immunotoxic effects of nanomedicinal products with regulatory immunotoxicity testing requirements

Figures & data

Figure 1 Nanoformulations in medicinal products.

Notes: Copyright ©2014. Dove Medical Press. Reproduced from Hafner A, Lovric J, Lakos GP, Pepic I. Nanotherapeutics in the EU: an overview on current state and future directions. Int J Nanomedicine. 2014;9:1005–1023.¹¹

Abbreviation: PEG, polyethylene glycol.

Table 1 Examples of immunotoxic effects reported after in vitro or in vivo testing of nanomaterials

Adverse side effects	Nanomaterial	Average size (nm)	Testing method	References
Inflammation due to ROS production	Nonporous silica nanoparticles	15	In vivo animal model Male SD rats Intravenous administration – single dose	^Citation137
ROS production	Carbon black	14	In vitro A549, CaCo, CaLu3, HaCaT, MDCK, MDCK2, RAW264.7, NIH3T3, NRK52E, and RLE6TN	^Citation62
ROS production and cell apoptosis	Titanium dioxide (TiO2) NPs	21	In vitro Beas-2B	^Citation138
NF-κB pathway activation Inflammatory cytokine secretion (TNF-alpha and IL-8)	Negatively charged PAA-AuNPs	–	In vitro THP-1 cells	^Citation65
Modification of cellular function NF-κB-regulated luciferase reporter induction	Citrate-stabilized AuNPs	10	In vitro Murine B-lymphocyte cell line (CH12.LX)	^Citation66
Improper antigen presenting cell maturation Partly exacerbate in vivo murine allergic airway inflammation by promotion of induction of T-helper immune responses	MWCNT	–	In vitro Bone marrow-derived DC In vivo ICR mice	^Citation67
Improper antigen-presenting cell maturation Exacerbate in vivo murine allergic airway inflammation by promotion of induction of T-helper immune responses	SWCNT	–	In vitro Bone marrow-derived DC In vivo ICR mice	^Citation68
Delayed-type hypersensitivity (DTH)	Resovist^® (Bayer AG, Leverkusen, Germany)	58.7	In vivo Male BALB/c mice Intravenous administration 1 hour before ovalbumin sensitization	^Citation70
Inflammatory responses via ROS, inflammasome, IL-1beta pathway	Titanium dioxide (TiO2) NPs	7–10	In vitro PBMC-derived DC	^Citation69
Th1/Th2 balance shift	Cerium oxide (CeO2)	3–5	In vitro PBMC-derived DC	^Citation69
Inflammation and oxidative stress	CNT	–	In vitro mϕ (rat alveolar mϕ [NR8383], human THP1-derived mϕ) Human microvascular endothelial cells Human epidermal keratinocytes Human bronchial epithelial (BEAS-2B) cells Human mesothelial (MeT-5A) cells Lymphoblastoid (MCL-5) cells	^Citation71
Generation of a pro-inflammatory and pro-oxidant environment in the lungs	CNT	–	Mice and rats	^Citation71
Immunosuppression	AgNPs	20 100	28-Day repeated dose Intravenous administration to Wistar rats	^Citation72, ^Citation73
TLR exploitation	PVM/MA NPs	230±5	In vitro Mouse DC from mouse femur bone marrow precursors In vivo C57/BL6 ip administration	^Citation75
Suppress the expression of TLR9 and altering of the CpG oligonucleotides Increase of TLR4-mediated LPS-induced production of pro-inflammatory cytokines	Titanium dioxide (TiO2) NPs Silicon dioxide (SiO2) NPs Zirconium dioxide (ZrO2) NPs Co NPs	TiO2 70 SiO2 15 ZrO2 5–30 Co 50–200	In vitro Human mϕ PMA-differentiated myelomonocytic U-937 cells	^Citation76
Post-translational citrullination of proteins	Silicon dioxide (SiO2) NPs Positively charged alumina-coated chloride ion-stabilized SiO2 NPs (Al-SiO2 NPs) Sc-SiO2 NPs SWCNT	SiO2 NPs 30, 80, and 400 Al-SiO2 NPs 40 Sc-SiO2 NPs 20 SWCNT: mean diameter 1–4 nm and length 1–3 µm	In vitro A549 and THP1 cells In vivo C57BL/6 mice – inhalatory exposure	^Citation78, ^Citation79
Generation of VEGFR2-mediated autophagy	Silica NPs (SiNPs)	62	In vivo ICR mice	^Citation81
Autophagic cell death through the Akt–TSC2–mTOR pathway in vitro Acute lung injury in vivo	PABS-, PEG-, or carboxylic acid (COOH)-functionalized SWCNT	–	In vitro Human lung adenocarcinoma A549 cells In vivo BALB/c mice	^Citation83
Potential autophagy due to excessive oxidative stress induction	AuNPs	20	In vitro MRC-5 human fetal lung fibroblast cells	^Citation82

Abbreviations: AgNPs, silver nanoparticles; AuNPs, gold nanoparticles; CNT, carbon nanotubes; DC, dendritic cells; DTH, delayed-type hypersensitivity; ip, intraperitoneal; mϕ, macrophages; MWCNT, multiwalled carbon nanotubes; NPs, nanoparticles; PAA, poly(acrylic acid); PABS, polyaminobenzene sulfonic acid; PBMC, peripheral blood mononuclear cell; PEG, polyethylene glycol; PVM/MA, poly(methyl vinyl ether-co-maleic anhydride); ROS, reactive oxygen species; Sc-SiO₂, sodium counterion-stabilized SiO₂; SiNPs, silica nanoparticles; SWCNT, single-walled carbon nanotubes; TLRs, toll-like receptors; ICR, Institute of Cancer Research; SD, Sprague Dawley; CpG, 5′-C-phoshate-G-3′; LPS, lipopolysaccharide; PMA, phorbol myristate acetate.

Figure 2 Illustration of the three pathways of complement activation-related pseudoallergy induction.

Notes: Republished with permission of ELSEVIER BV from Interactions of nanomaterials and biological systems: implications to personalized nanomedicine. Zhang XQ, Xu X, Bertrand N, Pridgen E, Swami A, Farokhzad OC. 2012;64(13); permission conveyed through Copyright Clearance Center, Inc.¹⁰⁵

Abbreviations: Ig, immunoglobulin; PEG, polyethylene glycol; SWCNT, single-walled carbon nanotubes; MBL, mannose binding lectin; MASPs, mannose-binding lectin-associated serin protease.

Table 2 Summary of the standard toxicity tests and the additional immunotoxicity assays recommended by the harmonized ICH S8 guideline

Standard toxicity studies

Hematology and clinical chemistry

Gross pathology and organ weights

Histology

Additional immunotoxicity studies

TDAR

Immunophenotyping

Natural killer cell activity assays

Host resistance studies

Macrophage/neutrophil function

Abbreviations: ICH, International Council for Harmonization of Technical Requirements for Pharmaceuticals for Human Use; TDAR, T-cell-dependent antibody response.

Table 3 Examples of immunotoxic effects of NMs or NMPs, and whether the testing methods included in the ICH S8 guideline are expected to detect the immunotoxic effects reported

Immunotoxic effects by NMs or NMPs	Testing techniques	ICH S8 guideline	Literature
Immunostimulation
Inflammasome activation (eg, CeO2 and SiO2 NMs)	In vivo studies	−	+
	In vitro studies	−	+
Inflammation (eg, Au NMs)	In vivo studies	+	+
	In vitro studies	−	+
Hypersensitivity
Lung hypersensitivity (eg, NiO and Co3O4	In vivo studies	−	+
	In vitro studies	−	+
CARPA
By liposomes, lipid-based NMs	In vivo studies	−	+
	In vitro studies	−	+
Immunosuppression
Reduction of IgG levels in vivo (eg, AgNMs)	In vivo studies	+	+
	In vitro studies	−	+
Myelosuppression (eg, PIBCA-conjugated doxorubicin and antimony and cobalt nanoparticles)	In vivo studies	−	+
	In vitro studies	−	+

Notes: Lung hypersensitivity refers to inhalatory exposure to NMs and also to NMPs. −, the absence to test on the adverse effects reported in literature; +, testing methods to detect these adverse immune effects.

Abbreviations: CARPA, complement activation-related pseudoallergy; ICH, International Council for Harmonization of Technical Requirements for Pharmaceuticals for Human Use; IgG, immunoglobulin G; NMs, nanomaterials; NMPs, nanomedicinal products; PIBCA, polyisobutylcyanoacrylate.

HafnerALovricJLakosGPPepicINanotherapeutics in the EU: an overview on current state and future directionsInt J Nanomedicine201491005102324600222

 PubMed Web of Science ®Google Scholar

ChenQXueYSunJKupffer cell-mediated hepatic injury induced by silica nanoparticles in vitro and in vivoInt J Nanomedicine201381129114023515466

 PubMed Web of Science ®Google Scholar

KrollADierkerCRommelCCytotoxicity screening of 23 engineered nanomaterials using a test matrix of ten cell lines and three different assaysPart Fibre Toxicol20118921345205

 PubMed Web of Science ®Google Scholar

ParkEJYiJChungKHRyuDYChoiJParkKOxidative stress and apoptosis induced by titanium dioxide nanoparticles in cultured BEAS-2B cellsToxicol Lett2008180322222918662754

 PubMed Web of Science ®Google Scholar

DengZJLiangMMonteiroMTothIMinchinRFNanoparticle-induced unfolding of fibrinogen promotes Mac-1 receptor activation and inflammationNat Nanotechnol201161394421170037

 PubMed Web of Science ®Google Scholar

SharmaMSalisburyRLMaurerEIHussainSMSulenticCEGold nanoparticles induce transcriptional activity of NF-κB in a B-lymphocyte cell lineNanoscale2013593747375623503581

 PubMedGoogle Scholar

InoueKKoikeEYanagisawaRHiranoSNishikawaMTakanoHEffects of multi-walled carbon nanotubes on a murine allergic airway inflammation modelToxicol Appl Pharmacol2009237330631619371758

 PubMed Web of Science ®Google Scholar

InoueKYanagisawaRKoikeENishikawaMTakanoHRepeated pulmonary exposure to single-walled carbon nanotubes exacerbates allergic inflammation of the airway: possible role of oxidative stressFree Radic Biol Med201048792493420093178

 PubMed Web of Science ®Google Scholar

ShenCCLiangHJWangCCLiaoMHJanTRIron oxide nanoparticles suppressed T helper 1 cell-mediated immunity in a murine model of delayed-type hypersensitivityInt J Nanomedicine201272729273722701318

 PubMed Web of Science ®Google Scholar

SchanenBCDasSReillyCMImmunomodulation and T helper TH(1)/TH(2) response polarization by CeO(2) and TiO(2) nanoparticlesPLoS One201385e6281623667525

 PubMed Web of Science ®Google Scholar

MollerPChristophersenDVJensenDMRole of oxidative stress in carbon nanotube-generated health effectsArch Toxicol201488111939196425212906

 PubMed Web of Science ®Google Scholar

De JongWHVan Der VenLTSleijffersASystemic and immunotoxicity of silver nanoparticles in an intravenous 28 days repeated dose toxicity study in ratsBiomaterials201334338333834323886731

 PubMed Web of Science ®Google Scholar

VandebrielRJTonkECMde la Fonteyne-BlankestijnLJImmunotoxicity of silver nanoparticles in an intravenous 28-day repeated-dose toxicity study in ratsParticle and Fibre Toxicology2014112124885556

 PubMed Web of Science ®Google Scholar

TamayoIIracheJMMansillaCOchoa-ReparazJLasarteJJGamazoCPoly(anhydride) nanoparticles act as active Th1 adjuvants through toll-like receptor exploitationClin Vaccine Immunol20101791356136220631332

 PubMed Web of Science ®Google Scholar

LucarelliMGattiAMSavarinoGInnate defence functions of macrophages can be biased by nano-sized ceramic and metallic particlesEur Cytokine Netw200415433934615627643

 PubMed Web of Science ®Google Scholar

MohamedBMVermaNKDaviesAMCitrullination of proteins: a common post-translational modification pathway induced by different nanoparticles in vitro and in vivoNanomedicine (Lond)2012781181119522625207

 PubMed Web of Science ®Google Scholar

VermaNKConroyJLyonsPEAutophagy induction by silver nanowires: a new aspect in the biocompatibility assessment of nanocomposite thin filmsToxicol Appl Pharmacol2012264345146122959926

 PubMed Web of Science ®Google Scholar

DuanJYuYYuYSilica nanoparticles enhance autophagic activity, disturb endothelial cell homeostasis and impair angiogenesisPart Fibre Toxicol2014115025266717

 PubMed Web of Science ®Google Scholar

LiuHLZhangYLYangNA functionalized single-walled carbon nanotubeinduced autophagic cell death in human lung cells through Akt–TSC2-mTOR signalingCell Death Dis20112e15921593791

 PubMed Web of Science ®Google Scholar

LiJJHartonoDOngCNBayBHYungLYAutophagy and oxidative stress associated with gold nanoparticlesBiomaterials201031235996600320466420

 PubMed Web of Science ®Google Scholar

ZhangXQXuXBertrandNPridgenESwamiAFarokhzadOCInteractions of nanomaterials and biological systems: implications to personalized nanomedicineAdv Drug Deliv Rev201264131363138422917779

 PubMed Web of Science ®Google Scholar