Assay Cascade

Characterisation stepCharacterisation step Type of characterisationShort description of testEUNCL identifier
Prescreening Size, Size distribution Dynamic light scattering (DLS) measures the hydrodynamic diameter of nanoparticles in solution EUNCL-PCC-01
Zeta Potential Zeta potential values provide an indirect measurement of the net charge on the nanoparticle (NP) surface. Among different approaches to characterize the superficial properties of NPs in liquid state, zeta potential measurement is one of the most accessible EUNCL-PCC-02
pH of nanoparticle suspensions The measurement of pH of nanoparticle suspensions in water based media will be performed in parallel to the other physical-chemical measurements in all the particular conditions tested, and pH values will be always associated with their outcomes. EUNCL-PCC-13
Endotoxin by kinetic turbidity LAL assay For the detection and quantification of Gram Negative Bacterial Endotoxin contamination each nanoparticle formulation will be examined using appropriate Limulus Amebocyte Lysate (LAL) assays including a positive product control.



Detection of Mycoplasma Samples may be subjected to testing for viable mycoplasma.  After incubation with a suitable indicator cell line which allows amplification of low-grade mycoplasma growth a PCR-based method is used for the detection of mycoplasma DNA encoding specific 16S rRNAs. EUNCL-STE-03
All nanomaterials will be analysed for the presence of microbial contamination. After spreading nanomaterials the on the surface of growth media and subsequent breeding microbial growth can be determined by visual colony detection and counting. EUNCL-STE-02.2
Drug loading Drug loading will be measured by dissolution of the NPs, and subsequent quantification of the drug by LC-MS/MS referenced against a pure drug standard. EUNCL-PCC-30
Detection of Gram Negative Endotoxin Detection and Quantification of Gram Negative Bacterial Endotoxin Contamination in Nanoparticle Formulations by Kinetic Turbidimetric LAL Assay. EUNCL-STE-001.2.1
PCC Cryo-TEM Cryo-TEM allows direct morphological visualization of nanomaterials/nanoparticles at near native state (e.g. physiological liquid media), reducing the risk of aggregation and of the introduction of artifacts due to sample preparation (e.g. evaporation can lead to aggregation of nanoparticles). The main advantage of using cryo-TEM is the possibility to observe the sample in its native state, since there is no need to use staining or fixation techniques EUNCL-PCC-20
Measuring NP aggregation propensities with DLS Nanoparticles (NP) tend to lose their colloidal stability and aggregate, if they are not properly stabilized. Generally speaking their aggregation propensity depends on temperature, time, pH and ionic strength of the dispersant/medium. These properties need to be checked in order to predict, how NP will behave when performing in vitro (or in vivo) studies. EUNCL-PCC-21
Size, Size Distribution In most cases, nanoparticle drug formulations are expected to avoid interaction with blood components as protein adsorption from blood might radically change the stability, fate and circulation time of nanoparticles, having effects on the bioavailability and pharmacokinetics of the carried or encapsulated drug molecules. DLS and MALS combined on-line with separation methods like Field-Flow Fractionation provide accurate size information for poly-dispersed samples in physiological media and for protein binding studies. EUNCL-PCC-22 
Free vs. encapsulated drug The free/bound drug ratio is determined after appropriate separation of the free dissolved drug from the carrier system (liposomes/polymeric) particles by using reverse phase HPLC with UV-VIS or fluorescent detector. EUNCL-PCC-31
Lipid composition Liposome composition will be measured by dissolution of the liposomes with organic solvent, and subsequent quantification of the lipid components by LC-MS/MS. Where applicable, lipid standards will be acquired from the sponsor; otherwise commercial standards will be used. EUNCL-PCC-32
Polymer composition   EUNCL-PCC-33
Drug release in complex media Drug release will be measured by the dual isotope labelling method (Skoczen et al, 2015). Quantification of free and bound drug will be by LC-MS/MS. EUNCL-PCC-035
Particle Tracking Analysis Measuring the Size and Concentration of Nanoparticles using Particle Tracking Analysis (PTA). EUNCL-PCC-023
In vitro immunology Hematolyic properties Erythrocytes comprise approximately 45% of whole blood by volume. Hemolysis refers to the damage of red blood cells leading to the release of erythrocyte intracellular content into blood plasma. When it occurs in vivo hemolysis can lead to anemia, jaundice and other pathological conditions, which may become life threatening. Hemoglobin is a dominant protein carried by erythrocytes. When it is contained inside the cell it plays a key role in carrying oxygen to other cells and tissues. However, extracellular hemoglobin is toxic and may affect vascular, myocardial, renal and central nervous system tissues. This is why all medical devices and drugs, which come in contact with blood are required to be tested for potential hemolytic properties. EUNCL-ITA-01
Platelet aggregation Platelets are small (~2 mm) anuclear cells obtained by fragmentation of megakaryocytes.  Platelets, also known as thrombocytes, play a key role in hemostasis.  Abnormal platelet counts and function may lead to either bleeding or thrombosis.  Assessing nanoparticle effects on human platelets in vitro allows for quick screening of their potential anticoagulant or thrombogenic properties mediated by direct effects on platelets.


Complement activation by EIA The complement system represents an innate arm of immune defense and is named so because it “complements” the antibody-mediated immune response.  Three major pathways leading to complement activation have been described: they are the classical pathway, alternative pathway and lectin pathway.  The classical pathway is activated by immune (antigen-antibody) complexes.  Activation of the alternative pathway is antibody independent. The lectin pathway is initiated by plasma protein mannose-binding lectin. EUNCL-ITA-005.2
Plasma coagulation times This assay assesses the effect a nanoparticle formulation may have on plasma coagulation time. Coagulation, i.e, blood clotting, is a highly complex process that involves many components. There are three main pathways for coagulation: intrinsic, extrinsic and the final common pathway. Extrinsic and intrinsic pathways converge into common pathway. Thrombin time (TT) is an indicator of the functionality of the final common pathway. EUNCL-ITA-12
Interaction with plasma proteins Proteins bind the surfaces of nanoparticles, and biological materials in general, immediately upon introduction of the materials into a physiological environment.  The further biological response of the body is influenced by the nanoparticle–protein complex.  The nanoparticle's composition and surface chemistry dictate the extent and specificity of protein binding.  In general, charged particles bind more proteins than their neutral counterparts.  Protein binding is one of the key elements that effect particle uptake by the phagocytic cells of the immune system, and therefore, the biodistribution of the nanoparticles throughout the body. Protein binding may or may not affect the activity of the protein in the “corona’ surrounding nanoparticle surface. As such using protein binding in lieu of specialized immunotoxicity assays is not recommended EUNCL-ITA-04
In vitro hematology Leukocyte proliferation This assay assesses the effect of a nanoparticle formulation on the basic immunologic function of human lymphocytes, i.e. measurement of lymphocyte proliferative responses. It will allow for measurement of a nanoparticles’ ability to induce proliferative response of human lymphocytes or to suppress that induced by phytohemaglutinin (PHA-M). EUNCL-ITA-006.2
Induced inflammatory cytokines Cytokine storm is a condition characterized by high plasma levels of inflammatory cytokines, chemokines and interferons which can be commonly induced by pathogens or their components (endotoxin, lipoproteins, DNA, RNA etc ). Cytokine storm can also be induced in response to certain drugs (e.g. recombinant proteins, therapeutic antibodies, macromolecular nucleic acid based therapeutics). Human whole blood and peripheral blood mononuclear cells (PBMC) are considered reliable and predictive model for this purpose. The data obtained from such in vitro studies is intended to supplement other preclinical data to create nanoparticle safety profile and ensure transition of nanomedicines toward clinical development. EUNCL-ITA-10
Leukocyte procoagulant activity Leukocyte procoagulant activity (PCA) is accepted as an important component in the onset of disseminated intravascular coagulation (DIC). DIC in cancer patients is often observed after initiation of therapy with cytotoxic oncology drugs that act by altering DNA replication (e.g., doxorubicin, daunorubicin, and vincristin). Cytotoxic oncology drugs acting by other mechanisms, (e.g., methotrexate and paclitaxel) do not induce DIC. DIC is also a common complication in sepsis. Cytotoxic drugs (doxorubicin, vincrisitn, and daunorubicin) and endotoxin have previously been shown to induce leukocyte PCA in vitro and DIC in vivo. In vitro, doxorubicin-induced leukocyte PCA has previously been linked to DIC in vivo. EUNCL-ITA-017
Determination of cytokine concentrations and Determination of cytokine concentration by flow cytometry  



In vitro toxicology LLC-PK1 Kidney cytotoxicity As part of the EU-NCL characterisation cascade all nanoformulations will be subjected to a cytotoxicity testing with in vitro cultivated cell lines. For this purpose, the porcine proximal tubule kidney cells (LLC-PK1) and human hepatocarcinoma cells (Hep G2), or other cell lines when necessary will be selected.  After exposure to the nanomaterial the cells will be tested for lactate dehydrogenase release as a measure of membrane integrity and cell death, and by using a tetrazolium dye that is reduced by live cells to a coloured formazan to measure the metabolic activity of cells. EUNCL-GAT-01
HepG2 hepatocarcinoma cytotoxicity Please refer to EUNCL_GTA-001 EU-NCL-GAT-02

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  • EUNCL at CRS Annual Meeting in Valencia
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  • Last General Assembly met in Stoos, Switzerland 23. - 24. May 2019
    The project’s last General Assembly took place under the motto ‘overview, impact and forecast’. The Work Package leader presented a pre-final report on their respective WPs, recapitulating the tasks accomplished during the last 3,5 years of the project’s runtime.

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