1. Knowledgebase of Nanomaterial-Biological Interactions and nano-TAB caBIG ICR F2F Stacey Harper, PhD Oregon State Sharon Gaheen, SAIC May 6, 2010

2. Agenda Knowledgebase of Nanomaterial-Biological Interactions (NBI) nano-TAB

3. Nanomaterial-Biological Interactions Knowledgebase Stacey Lynn Harper, Ph.D. Environmental and Molecular Toxicology Chemical, Biological and Environmental Engineering

4. Common Need To Understand the Governing Factors in Nanomaterial-Biological Interactions Biomedical Applications Nanomaterial therapeutics Biomimicry Diagnostics and imagingProsthetics Targeted drug deliveryBionics Regenerative medicine Inform Regulatory Agencies Risk assessment Improve public perception Safer Nanomaterials Biological effects from exposure Industry feedback on toxic potential Material modifications that minimize hazard

5. Backcasting to the Future Backcasting Present Future VISION Does it move us in the right direction?

6. A Priori Forecasting of Biological and Environmental Effects of Nanomaterial Exposure Vision

7. Backcasting to the Future Backcasting Present Future VISION Organize and integrate data (repositories) Define nanomaterial commonality Employ weight-of-the-evidence techniques Define nanoSARs Identify design rules

8. Nanomaterial Commonality Relative importance of nanomaterial characteristics unknown Complexity Requires Informatics Solutions

9. Physicochemical Properties Chemicals Structure pKa Solubility log P 3-D Molecular Structure 3-D Crystal Structure Illustrations reproduced with permission from Herr’s Carbon Fullerene Gallery http://www.vincentherr.com/cf/nanomain.html http://www.vincentherr.com/cf/nanomain.html Nanomaterials Chemical Structure Core Particle Composition Size Shape Charge Surface Chemistry Surface Area Agglomeration State Zeta Potential

10. Nanomaterial Commonality Relative importance of nanomaterial characteristics unknown Nanomaterials are diverse Complexity Requires Informatics Solutions

11. 3 rd Dimension of Periodic Table Size & geometry

12. Diversity of Zinc Oxide Nanoparticles Photos adapted: Dr. Z Wang, Georgia Tech

13. Complexity Requires Informatics Solutions Shaw et al., 2008. Perturbational profiling of nanomaterial biologic activity. PNAS, 105(21): 7387–7392 Profiling the biological activity of nanoparticles

14. Complexity Requires Informatics Solutions Shaw et al., 2008. Perturbational profiling of nanomaterial biologic activity. PNAS, 105(21): 7387–7392 Comparing nanoparticle activity profiles

15. Complexity Requires Informatics Solutions Shaw et al., 2008. Perturbational profiling of nanomaterial biologic activity. PNAS, 105(21): 7387–7392 Hierarchical clustering of nanoparticles based on activity

16. Nanomaterial Commonality Relative importance of nanomaterial characteristics unknown Nanomaterials are diverse Necessary to have complex nanomaterial descriptors rather than simplified (SMILES) Complexity Requires Informatics Solutions C[C@@](C)(O1)C[C@@H](O)[C@@]1(O2)[C@@H](C)[C@@H]3CC=C4[C@]3(C2)C(=O)C[C@H ]5[C@H]4CC[C@@H](C6)[C@]5(C)Cc(n7)c6nc(C[C@@]89(C))c7C[C@@H]8CC[C@@H]%10[C @@H]9C[C@@H](O)[C@@]%11(C)C%10=C[C@H](O%12)[C@]%11(O)[C@H](C)[C@]%12(O%1 3)[C@H](O)C[C@@]%13(C)CO Caphalostatine-1

17. Nanomaterial Commonality Relative importance of nanomaterial characteristics unknown Nanomaterials are diverse Necessary to have complex nanomaterial descriptors rather than simplified (SMILES) Complexity Requires Informatics Solutions Collaboratory of Structural Nanobiology

18. Nanomaterial-Biological Interactions Knowledgebase

19. Interoperable, Federated System of Data/Knowledgebases for Nano-Bio Informatics Data Repositories NTP (NIEHS) NCL (NCI, FDA, NIST) NBI (ONAMI) NIL (NIOSH)

20. Agenda Knowledgebase of Nanomaterial-Biological Interactions (NBI) nano-TAB

21. Goal To develop a specification to facilitate the import/export of nanomaterials and their characterizations to/from nanotechnology resources

22. Specification Development Challenges Nanotechnology studies involve numerous assays with measurements dependent on the protocol and technology types Nanotechnology protocols lack standardization Capturing the assay conditions in a standardized fashion is imperative to understand the polydisperse nature of the nanomaterial Structure-Activity-Relationships (SARs) are important as the nanomaterial structure has a direct impact on biological activity. There are minimal standards supporting nanomaterial structures and models. Standardize Manufacture

23. Nanotechnology Assays and Technology Types Wash U NanoParticle Ontology (NPO) v. 2010-04-30 AssaysTechnology Types

24. Nano-TAB Approach Leverage and extend the Investigation / Study / Assay (ISA- TAB) specification Utilize and/or extend concepts from the LS DAM nano-subdomain Use the NPO as an ontology term source Draft example files using existing characterization studies as a validation of the specification NCL Dendrimer-Based MRI Contrast Agents NBI Exposure Study Investigation Study Assay Data Files Material Study nano-TAB Structure Structure

25. LS DAM Nano-Subdomain Material Experiment Protocol Study

26. NCL Dendrimer-Based MRI Contrast Agents Investigation Objective Characterize a PAMAM [Poly(amidoamine)] dendrimer with an associated gadolinium chelate MRI contrast agent Studies Physico-Chemical Measure the size of the dendrimer using dynamic light scattering (DLS) and fractionation methods and analyze the effects of sample concentration, buffer, and temperature on particle size (stability) Analyze purity by HPLC, capillary electrophoresis, and fractionation methods Obtain molecular weight using MALDI-TOF mass spectrometry and fractionation methods Use inductively coupled plasma optical emission spectroscopy and a 3T clinical MRI machine to determine the relaxivity of the sample as compared with free Magnevist Immunotoxicology Analyze nanoparticle blood contact properties and the effects of coagulation pathways on the integrity of blood cellular components including hemolysis and platelet aggregation Evaluate the effects on in vitro immune function including phagocytic update, leukocyte prolifertion, oxidative burst or chemotaxis In Vitro Toxicology Evaluate biocompatibility leveraging porcine renal proximinal tubule cells (LLC-PK1) and human hepatocarcinoma (HEP-G2) cell lines to determine cytoxicity under diverse experimental conditions

27. NCL Dendrimer-Based MRI Contrast Agents DRAFT Investigation File (1 of 2) Investigation Ontology References Publications Contacts Study Study Design Study Publications Study Factors Study Assays

28. NCL Dendrimer-Based MRI Contrast Agents DRAFT Investigation File (2 of 2) Study Protocols Additional Studies

29. NCL Dendrimer-Based MRI Contrast Agents Study Samples NCL20 – G4 tris (hydroxyl) terminated PAMAM dendrimer NCL21 – G4 pyrrolidinone terminated PAMAM dendrimer NCL22 - G4.5 COONa terminated PAMAM dendrimer NCL23 - G4.5 COONa terminated PAMAM dendrimer-Magnevist® complex (Primary Nanomaterial) NCL24 - Commercially available Magnevist® (NCL24) was used as a control NCL25 - G4 tris (hydroxyl) terminated PAMAM dendrimer-Magnevist® complex NCL26 - G4 pyrrolidinone terminated PAMAM dendrimer-Magnevist® complex Study Samples Physico-Chemical – All Samples Immunotoxicology – NCL22, NCL23, NCL24 In Vitro Toxicology – NCL22, NCL23, NCL24 Porcine renal proximinal tubule cells (LLC-PK1) and human hepatocarcinoma (HEP-G2) cell lines

30. NCL Dendrimer-Based MRI Contrast Agents Draft Study Files Physico-Chemical Immunotoxicology/In Vitro Toxicology Sample ReferencesMaterial Characteristics Cell Types

31. NCL Dendrimer-Based MRI Contrast Agents Material

32. NCL Dendrimer-Based MRI Contrast Agents Draft Material File Sample Material Contacts Material Files Material Components Material Characteristics Material Associations

33. NCL Dendrimer-Based MRI Contrast Agents Assays Hydrodynamic Size/Size Distribution via Dynamic Light Scattering (DLS) Thermal Stability of NCL22 Hydrodynamic Size Comparison of NCL22 and NCL23 Effect of Hydrodynamic Size on NCL23 Concentration in PBS Particle Number Density and Mean Inter-particle Distance Gadolinium Quantitation via Inductively Coupled Plasma Optical Emission Spectroscopy (ICP-OES) Molecular Weight Measurement by Mass Spectrometry UV-Vis Spectra Reverse Phase HPLC Capillary Electrophoresis (CE) Size Exclusion Chromatography (SEC) – Multiple Angle Laser Light Scattering (MALLS) MRI Relaxivity Measurements for NCL 23 (in vitro) Asymmetrical Flow Field-Flow Fractionation (AFFF) – Multi Angle Light Scattering (MALS) MRI Relaxivity Measurements for NCL23 (in vitro) MTT Cytotoxicity Assay in LLC-PK1 Cells (GTA-1) LDH Cytotoxicity Assay in LLC-PK1 Cells (GTA-1) MTT Cytotoxicity Assay in Hep-G2 Cells (GTA-2) LDH Cytotoxicity Assay in Hep-G2 Cells (GTA-2) Endotoxin Spike Recovery in the Presence of Dendrimer Particles (STE-1) Microbial Sterility Test (STE-2) Mycoplasma Contamination Test (STE ‑ 3) Nanoparticle Hemolytic Properties (ITA-1) Nanoparticle Ability to Induce Platelet Aggregation (ITA-2) Nanoparticle Toxicity to Bone Marrow Cells (ITA-3) Nanoparticle Effect on Coagulation (ITA-12) Interaction with Plasma Proteins (ITA-4) Complement Activation (ITA-5) Nanoparticle Effect on Leuckocyte Proliferation (ITA-6) Nitric Oxide Production by Macrophages (ITA-7) Nanoparticle Effect on Chemotaxis (ITA-8) Phagocytosis Assay (ITA-9) Cytokine Secretion by PBMC (ITA-10) Potential NCL22 Effects on LPS-Induced Cytokine Secretion by PBMC (ITA-10) Cytotoxic Activity of NK Cells by 51Cr-release Assay (ITA- 11A) Cytotoxic Activity of NK Cells by Real-Time Cell Electronic Sensing (RT-CES) Physico-Chemical In Vitro Toxicology Immunotoxicology

34. NCL Dendrimer-Based MRI Contrast Agents Draft Assay File (1 of 2) SampleProtocol AssayAssay Measurements

35. NCL Dendrimer-Based MRI Contrast Agents Draft Assay File (2 of 2) Assay MeasurementsAssay Files

36. NCL Dendrimer-Based MRI Contrast Agents Structure Use of PDB format to represent structural information in a compatible format Use of the minimum coordinate specification in PDB format Consider use of MOL2 format for descriptive information (atomic point charges) PDB and MOL2 formats can be converted into a variety of other formats using OpenBabel Use of standard formats will ensure compatibility with existing visualization software

37. NCL Dendrimer-Based MRI Contrast Agents Draft structure File (PDB) X, Y, Z Coordinates Header Compound

38. Nano-TAB Status and Next Steps Drafted example files for physico-chemical (size) characterization from the NCL Dendrimer investigation and NBI exposure study Plan to obtain feedback from other data sources including the National Toxicology Program (NTP) Add concepts to the NPO, if needed, in support of nano-TAB Assay names and technology types under curation Develop a collaboration with ISA-TAB and resolve open questions Common nomenclature for sample and material identifiers Level of granularity for representing studies and assays Provide/obtain recommendations to/from the LS DAM team in support of nano-TAB concepts Investigation, Study, Sample, Assays, Protocols Draft the nano-TAB specification and obtain feedback from the nanotechnology community

39. References nano-TAB http://gforge.nci.nih.gov/docman/index.p hp?group_id=69&selected_doc_group_i d=5653&language_id=1http://gforge.nci.nih.gov/docman/index.p hp?group_id=69&selected_doc_group_i d=5653&language_id=1 LS DAM Nano Subdomain https://gforge.nci.nih.gov/plugins/scmsvn /viewcvs.php/*checkout*/requirements/L SDAM%20R1_2.EAP?root=lsdamhttps://gforge.nci.nih.gov/plugins/scmsvn /viewcvs.php/*checkout*/requirements/L SDAM%20R1_2.EAP?root=lsdam caBIG ICR Nano Data Standards Document http://sites.google.com/site/cabignanowg /data-sharing-and-nanotechnology- standards/data-sharing-standardshttp://sites.google.com/site/cabignanowg /data-sharing-and-nanotechnology- standards/data-sharing-standards MAGE-TAB http://www.mged.org/mage-tab/MAGE- TABv1.0.pdfhttp://www.mged.org/mage-tab/MAGE- TABv1.0.pdf http://www.mged.org/mage- tab/spec1.0.htmlhttp://www.mged.org/mage- tab/spec1.0.html ISA-TAB http://isatab.sourceforge.net/ Nano-TAB Project Team Juli Klemm, NCI CBIIT Stacey Harper, Oregon State Sharon Gaheen, SAIC Sue Pan, SAIC Nathan Baker, Wash U Dennis Thomas, Wash U David Paik, Stanford Grace Stafford, JAX Labs Raul Cachau, NCL Marty Fritts, NCL Liz Hahn-Dantona, Lockheed