Presentation to

ASTM E56 Workshop

May 19, 2005

Scott McNeil, Ph.D.

Director, Nanotech Characterization Lab
SAIC-Frederick

1
 Outline

• Background
• NCL
• Assay Cascade
– Physical Characterization (Dr. Patri)
– In vitro and in vivo Pharm/Tox (Dr. Stern)

2
 Why Nano?

Therapeutic Benefits
• Solubility
Multi-functional Nanoparticles
– Carrier for hydrophobic
entities Drug
Contrast agent
(X-ray, MRI)

• Multifunctional capability
delivery
indicator

• Active and passive Cell

death

targeting sensor

– Ligands; size exclusion Cancer cell

targeting

• Reduced toxicity Therapeutic

Jim Baker, University of Michigan

3
 Nanotech at NCI

Background
• NCI has funded exploratory work over the past 6
years on integrating nanotechnology into
biomedical research
• Unconventional Innovations Program (UIP)
– Diagnostics (Imaging)
– Therapeutics
• Priority is to now transition that research into the
clinical realm.

4
•Run by Office of Technology and Industrial Relations
(OTIR)
•Director: Dr. Greg Downing
•Extramural Budget: $144M over 5 years
•Launched on Sept 13th, 2004
•Website: [Link]

•Consensus among cancer researchers that significant

obstacles must be overcome in order to transition ‘nano’
to clinical realm
•Critical lack of available standards
•1st principles characterization
•Regulatory uncertainty
5
 The Nanotechnology
Characterization Laboratory

Mission Statement
• The Nanotechnology Characterization
Laboratory (NCL) will perform and standardize
the pre-clinical characterization of nanomaterials
developed by researchers from academia,
government, and industry.
• The NCL will serve as a national resource and
knowledge base for cancer researchers, and
facilitate regulatory review and translation of
nanomaterials and devices into the clinical
realm.

6
 NCL Objectives

• Identify and characterize critical parameters related

to nanomaterials’ biocompatibility; structure-activity
relationships.

• Establish and standardize an assay cascade for

nanomaterial characterization.

• Examine the biological characteristics of multi-

component/combinatorial platforms.

• Engage and facilitate academic and industrial-based

education and knowledge sharing.
7
 The NCL

The Nanotechnology Characterization

Laboratory
– Provides critical infrastructure support for
Alliance
– Performs pre-clinical characterization of
nanomaterials intended for cancer therapeutics
and diagnostics
– Collaboration between NCI, NIST, US FDA

8
 NCL Concept of
Operations

Sources of
Nanomaterials
Cancer
Centers of
Nanotech NIS
Excellence T Detection
(CCNEs)
Academia

Big Pharm Physical

Characterization
Small
Business Diagnostics
In
NCI, NIH, NSF Vitro
Grants
NCL In
DoD, DoE Vivo
Unconventiona
Therapeutic
l Innovative
s
Program (UIP)

NCL conducts pre-clinical characterization in support

of an Investigative New Drug (IND) submission to the
FDA 9
 NCL Assay Cascade

Physical
Characterization:
– Size In Vitro:
– Size distribution – Binding
– Molecular weight – Pharmacology In Vivo:
– Density – Blood contact – Absorption
– Surface area properties – Pharmacokinetics
– Porosity – Cellular uptake – Serum half-life
– In vitro absorption, – Protein binding
– Hydrophilicity
distribution, –
– Surface charge density Tissue distribution
metabolism,
– Purity excretion – Metabolism
– Sterility – Excretion
– Surface chemistry
– Stability 10
In Vitro Cascade

11
 In Vitro
Example: Hemolysis Protocol

NCL Method ITA-1

Analysis of Hemolytic Properties of

Nanoparticles 120
Nanotechnology Characterization laboratory
National Cancer Institute at Frederick 100
SAIC-Frederick ASTM recommended
Frederick, MD 21702 positive control
(301)-846-6939 80 (polynitrile)
% Hemolysis
60

40

20

0
1 2 3 4 5
0.000 19.8 0.000 101 0.000
Sample
1-NC (polyethylene); 2- PC (nitrile); 3- dH2O; 4 - 50nm polystyrene
NP; 5- silica coated glass beads 12
 Physicochemical Characterization
and Standardization of Nanoparticles

Presentation to
ASTM E56 Workshop
May 19, 2005

Anil K. Patri, Ph.D.

Senior Scientist, Nanotech Characterization Lab
SAIC-Frederick

13
 NCL Objectives

• Identify and characterize critical parameters related

to nanomaterials’ biocompatibility; structure-activity
relationships.

• Establish and standardize an assay cascade for

nanomaterial characterization.

• Examine the biological characteristics of multi-

component/combinatorial platforms.

• Engage and facilitate academic and industrial-based

education and knowledge sharing.

14
 NCL Assay Cascade

Physical
Characterization:
– Size In vitro:
– Size distribution – Binding
– Molecular weight – Pharmacology In vivo:
– Density – Absorption
– Blood contact
– Surface area properties – Pharmacokinetics
– Porosity – Cellular uptake – Serum half-life
– Hydrophilicity – Protein binding
– Toxicity
– Surface charge density – Tissue distribution
– Purity – Metabolism
– Sterility – Excretion
– Surface chemistry
– Stability 15
 Nanoparticles by type
for medical applications
HN H2 N
H2 N H N 2 H2 N
H2 N 2 H2N H2N H2N H2N
H2 N H2N H2N H2N H N
H2 N NH NH H2N H N 2
H2 N NH NH NH 2
H2 N NH O
H2 N NH O O NH NH
NH O O NH NH O NH NH H2N
H2 N NH O O O O NH NH H2N
O O NH
NH
NH O O O NH O O
O O N N NH2
H2 N NH O N NH2
H2 N O O NH
N N N NH NH2
NHO N N N
O
H2 N NH HN N O NH NH2
NH N
H2 N O O O NH
NH NHHN O NH HN
H2 N O O NH N O NH

• Organic Nanoparticles (e.g.: Polymers, Dendrimers)

NH NH NH2
H2 N NH NHHN O O O HN O O NH
O N O NH2
O N HN N
NH O
H2 N O O O O
N N N NH
H2 N NHO O O NH2
HN N N O NH NH
N N HN N NH2
NH NH O
H2 N O NH HN
O N O NH
NH O NH2
H2 N N O O NH HN O
NHO HN O HN NH N NH NH2
O O O
NH N H O O O
H N N HN O NH N NH
H2 N N O N O
H NH NH2
H2 N H O N N
NO N O N O NH2
O N N O
N HN HN N NH
O NH O
O O
H2 N H NH O NH NH NH
N HN O 2
H O NH HN H N
H2 N N N N NH2
NO O NH N NH O
O NH
N O O O
H O HN O
O N NH
H2 N N O O NH2
N N N
H
N O HH N HN
N
O O NH2
H2 N N N H O O NH
O N O NH O
H N H O NH NH2
O N NH H N
H2 N N N O NH
H N N
H H N O NH2
N O O H N N O NH
H2 N N O H
N O O O
O N N N NH NH
H H N NH N H O N
O N H
H2 N N
HN O O N N N NH2
H O N H
N O NH O O
H2 N N O H NH2
N N O
N O O N
OO H
O O N N NH2
H2N N O N O N
N O O NH H H
H HN O NH2
H2N N
N O N H H
H H N O HN N
NH O
O N O N
N O O H N O N NH2
H2N N H O N N O
N O O N N N H H
H H N H NH2
H N N
H2N N N N O
O N H N H
H N H O N O
O N O
O H N N NH2
H2N N N H O N O N
N O HH N NH HH H
H HN N
N O O N NH2
H2N N O O N O

• Inorganic Nanoparticles (e.g.: Iron oxide, gold nanoparticles)

H O O N
N O H
N HN
NH N O O N
N O O N N NH2
H2N N O H
H O NH
HN O N NH2
H2N N O O HN H
H NH O
HN N O
O N NH NH NH
HN HN O
O O N O N
H2N N O N O N O H NH2
H O N
N N O N NH2
H2N O N N N N H
H N H N HN
NH N H O OHN
NHO HN O
O HN O O NH O NH
N
H2N N O O O HN NH2
NH O O
O HN HN O NH2
H2N O NH O NH NH O HN
NH N HN N N
N NH
H2N HN OHN NH2
NHO N N
O N N O O NH2
H2N O O HN
NH O N O
O N N
O N O HN O HN NH2
H2N NH NHHN O NH HN
NH O O
H2N O O O HN NH2
H2N
NH
O NHHN O O O NHHN N O NH2
NH N NH HN HN
H2N NH HN N NH2
NHO N N OHN
H2N N O NH2
O N O
NH O N HN NH2
H2N N N N O O
NH O O N HN
NH O O HN
H2N O O HN HN NH2
NH NH O O
O O O O O HN NH2
H2N NH O O O HN NH2
H2N NH HN HN
NH NH NH HN NH2 NH
H2N H2N NH HN HN 2
NH NH2
H2N NH2
H2N NH2
H2N H2N H2N NH2 NH2
NH2
H2N H2N NH2

• Organic/Inorganic hybrids (e.g.: Nanocomposites, core-shell type, Gd-chelates)

• Carbon based (e.g.: Functionalized fullerenes)

• Liposomes (e.g.: Functionalized, inclusion complexes)

• Nanoemulsions (e.g.: Oil-water-surfactant mixtures)

• Biological nanoparticles (e.g.: Protein and peptide based nanoparticles with

other biological components)

Terminology and Nomenclature will be addressed by E56 sub-committee

16
 Core parameters to define
physicochemical property of material
O

O
O OH
O O

N H James Baker
H
OH O University of Michigan
H
HO O
O
O O

Small molecules Nanomaterial

• Elemental analysis  Composition • Microscopy (AFM,

• Mass  Physical properties TEM, SEM)
• NMR  Chemical properties • Light scattering (Static,
• UV-Vis  Identification Dynamic)
• IR  Quality • SEC, FFF
• HPLC  Purity • Electrophoresis (CE,
• GC  Stability PAGE)
• Polarimetry • Zeta sizer
• Fluorimetry

Same parameters – different characterization methods

17
 Standardization of Characterization methods

Property Instrumentation

Size, Size Distribution, Topology Light Scattering, Microscopy (SEM, TEM,

AFM), SANS, SAXS

Purity, Composition, MW HPLC, SEC, NMR, Mass Spectrometry,

Light scattering, Atomic Absorption
and Emission, PAGE, CE

Functionality, Charge NMR, FTIR, UV-Vis, Fluorimetry, PAGE,

Zeta potential Zeta sizer, CE, pH titrations

Stability, Reproducibility Chemical analysis, above methods

Physical characterization in collaboration with NIST with input from

academia, industry and government agencies

All properties of the nanomaterial should be defined for standardization

18
 Size, size distribution
with microscopy
Visualization of Poly(amidoamine)
(PAMAM) Dendrimers by TEM AFM

G 10 G9

Gold colloids

G8 G7

G6 G5
Functionalized
gold particles

C.L. Jackson, H.D. Chanzy, F.P. Booy, B.J. Drake, D.A.

Tomalia, B.J. Bauer, E.J. Amis, Macromolecules, 31(18),
6259, 1998 19
 Molecular weight determination

Mass spectrometry Light scattering

• Small organic molecules • Ideal for nanomaterial
• Peptides and proteins • Gentle method
• Low [Link]. polymers • Size, size distribution
• Lower generation dendrimers • Hydrodynamic size
• Batch mode
 Not suitable for very high • Flow mode
molecular weight material

20
 Flow mode analysis
of Nanoparticles

ORGANIC AND CARBON ORGANIC-INORGANIC HYBRIDS,

BASED NANOPARTICLES LIPOSOMES, METAL NPs

Liquid chromatography
Field Flow Fractionation (FFF)
(HPLC/SEC)

UV-Vis, Fluorescence
Refractive Index
Light Scattering (MALLS, DLS)

Molecular Size/ Size Stability Hydrodynamic Monodispersity

Weight Distribution Aggregation Radius Purity
21
 Quality, Purity, Functionality

UV-Vis Spectroscopy Infrared spectroscopy

• Qualitative and quantitative analysis • Functional group analysis
• Kinetic studies • Surface characteristics
• Stability studies

NMR spectroscopy Electrophoresis

CE and PAGE
• 1H and 13C NMR
• Identity • Separation based on charge/mass
• Purity • Purity
• Functionality • Component distribution
• Quantitation from integration

22
 Multifunctional Nanomaterial

James Baker, University of Michigan

 Targeting, Imaging, therapeutic, reporter functions

 Complex nanomaterial for analysis

 Control particles with each component or control components

shall be established and characterized
 Reproducibility
– authenticated by physical, in vitro and in vivo test results

 Analysis to confirm the homogeneity in ligand distribution (e.g.: CE)

 Limitations in physical and chemical characterizations will be defined

 Detection limit of impurities or free individual components in the preparation

23
NCL Pharmacology and Toxicology
Preclinical Protocols

Presentation to
ASTM E56 Workshop
May 19, 2005

• Stephan Stern, Ph.D.

• Scientist, Nanotech Characterization Lab
• SAIC-Frederick

24
 NCL Pharmacology and Toxicology
Preclinical Protocols

Outline

I. In Vitro Pharmacology and Toxicology

II. In Vivo Pharmacokinetics

III. In Vivo Toxicology

25
 NCL Pharmacology and Toxicology
Preclinical Protocols

Objectives of NCL Preclinical Testing Program

• Develop Standardized Assay Cascade for

Nanoparticle Characterization

ASTM Guidance

ASTM F 1904-98: Standard practice for testing the Biological

Responses to Particles in vivo
ASTM F 1903 – 98: Standard Practice for Biological Responses to
Particles in vitro
26
 NCL Nanotherapeutics and
Diagnostics

Nanoparticle Applications

• Cytotoxic oncology drugs

– ex. dendrimer/cytotoxic drug
formulation, liposome/cytotoxic drug
formulation

• Imaging/Diagnostic Agents
– ex. Denrimer/gadolinium MRI agent,
Quantum dot diagnostic agent

27
 NP Structure-Activity
Relationship

I. Surface hydrophobicity
- Hydrophobic surface- taken up by RES system
- Hydrophilic surface- Increased systemic half-
life, enhanced permeability and retention in tumors
(EPR)

II. Surface Charge

- Toxicity: cationic>anionic>neutral

III. Surface reactivity

- More reactive (↑ROS)= ↑Toxicity 28

 NP Structure-Activity
Relationship

IV. Size (aggregation/disaggregation tendency)

- Lung deposition
(titanium dioxide, Oberdorster et al., 1994)
- Biodistribution (dendrimers, Malik et al., 2000
- Clearance mechanisms (i.e. renal, biliary)
- Subcellular distribution
e.g. < 9 nm access nucleus via nuclear pores
(QD, Lovri et al., 2005)
- Particle surface area/cellular interaction
29
 Mechanisms of Nanoparticle
Toxicity

Toxic mechanisms attributed to NP’s

• Oxidative stress
(fullerenes, Oberdorster 2004; polystyrene, Fernandez-
Urrusuno et al., 1997)
• Induction of apoptosis
(cationic dendrimers, Kuo et al., 2005)
• Protein binding (quantum dots, Lovri et al., 2004)
• Macrophage dysfunction (liposomal doxorubicin,
Daemen et al., 1995; titanium dioxide, Renwick et al.,
2001) 30
 Oxidative Stress

(1) ROS Generation

2GSH _
NADP+ O2• O2 + e-
GR
NADPH + H + 2H+
_
O2• SOD
GSSG + H2O
GSHPx Fullerene

GSH H2O2 + O2
(2) Loss of GSH Homeostasis
H2O2
Fe++ CAT
_
O2•
_ 2 H2O + O2
O2 + OH + OH •

(3) Cellular Damage Lipid Peroxidation Protein/DNA31Damage

 In Vitro Protocols

Study Cell line Parameter Specific Assay

Rat/human hepatic Oxidative/Conjugative in vitro
microsomes Metabolic stability
metabolism

Leukemia-(HL60)
Pharmacology Non Small Cell Lung (NCI- Efficacy-Necrosis (Cytotoxicity) MTT Assay
H460)
Breast-MCF7
CNS-SF268 Efficacy-Apoptosis (Cytotoxicity) Caspase-3 activation

Rat hepatic primaries MTT Assay (viability), LDH

(Sprague Dawley) Necrosis
(membrane integrity)

Apoptosis Caspase-3 activation

GSH homeostasis (DTNB assay),

Oxidative Stress
lipid peroxidation (TBAR assay)

Toxicology CYP 1A1 (EROD), CYP 1A2

Cytochrome P450 induction
(MROD), CYP 2B1 (PROD)

Pig renal proximal tubular

cells (LLC-PK1) MTT Assay (viability), LDH Assay
Necrosis (membrane integrity), -GT assay
(membrane integrity)

Apoptosis Caspase-3 activation

32
 In Vivo PK and Toxicity Studies

Rats are the preferred rodent species :

-Large blood volume for serial blood sampling

-Predictive of human toxicities (HTs)

(Olson et al., 2000)

33
 In Vivo Pharmacokinetics

• Single/repeat-dose PK/TK/tissue distribution

• Clinical Tx cycle
-Schedule
-Duration
-Route
-Formulation

• Quantitation method
-radiolabeled nanoparticle (Scintillation)
-Imaging
-ELISA

• PK Parameters
-AUC, Cmax, CL, t ½, tmax

Based on FDA Pre-clinical Guidance

34
 In Vivo Pharmacokinetics

Time
Purpose Duration Point’s Groups Tests Comments

Dosing, blood
draws by
Plasma PK profile/
scintillation counting of Jugular
Tissue distribution 1X, 10X (5 F SD Rats/Tx)
24 hrs 8 plasma and tissue catheter,
(Liver, lungs, kidney, heart,
samples (NCL) cardiac
spleen brain)
puncture (final
tp)

35
 In Vivo Toxicology Studies

• Single/Repeat-Dose Acute/Subacute Toxicity

-Rats (determine STD10/NOAEL/Lethal dose)

• Clinical Tx Cycle
-Schedule
-Duration
-Route
-Formulation

• Endpoints monitored
-Hematology
-Clinical chemistry
-Gross pathology
-Histopathology
-Clinical signs
Based on NCI DTP toxicology protocols 36
 In Vivo Toxicology Studies

Dose Range-Finding Toxicity Study

Purpose Duration Groups Tests Comments
BW
measured
Clinical daily,
determine
chemistries, euthanasia
dose at
ctrl, 10X, 50X, 100X histopathology, criteria
which 14 days
(5 M+F SD Rats/Tx) hematology, gross
toxicity is (decrease
pathology, clinical
observed in body
observation (PHL)
weight ≥
20%)

37
 Comprehensive Toxicology

Histopathology

Brain Pancreas Salivary gland

Lymph node Esophagus Parathyroid
Thyroid Trachea Adrenal
Pituitary Heart Kidney
Thymus Gall Bladder Liver
Spleen Lung Duodenum
Ileum Rectum Stomach
Cecum Colon Jejunum
Lymph node Epididymis Ovary
Prostate Seminal vesicle Testis
Urinary bladder Uterus Eye
Hardian gland Nasal Sections Femur
Femur Vertebra Spinal cord
Mammary gland Skin/Subcutis Tongue

38
 Comprehensive Toxicology

Hematology

Erythrocyte count (RBC)

Hemoglobin (HGB)
Hematocrit (HCT)
Mean corpuscular volume (MCV)
Mean corpuscular hemoglobin (MCH)
Mean corpuscular hemoglobin concentration (MCHC)
Platelet count (Plate)
Reticulocyte count (RETIC)
Total leukocyte count (WBC)
Differential leukocyte count
Nucleated red blood cell count
39
 Comprehensive Toxicology

Clinical Chemistry

BUN AST ALT GGT

GLUC Creatinine total protein Albumin
Globulin A/G Sodium Potassium
Chloride

40
 Environmental Aspects

Studies Applicable to Environmental Risk Assessment

• General Cytotoxicity Assays- determining concentration-

response relationships.

• Mechanistic Studies- Identifying apoptosis, oxidative stress

and cytochrome P450 induction/suppression as potential
mechanisms

• In Vivo Toxicology Studies- Identification of target organs

• General ADME- define t1/2, clearance mechanisms (i.e.

metabolism, biliary excretion, renal clearance, etc.)
41
 NCL Pharmacology and
Toxicology
Preclinical Standards

References

1 DeGeorge et al. (1998) Regulatory considerations for preclinical development of anticancer

drugs. Cancer Chemother Pharmacol 41: 173-185.
2 Guideline for Industry: Pharmacokinetics: Guidance for Repeated Dose Tissue Distribution
Studies, FDA:CDER: March 1995
3 Guidline for Industry: Toxicokinetics: The Assessment of Systemic Exposure in Toxicity
Studies, ICH:S3A:March 1995
4 Guidance for Industry: S6 Preclinical Safety Evaluation of Biotechnology-Derived
Pharmaceuticals
5 Guidance for Industry: Single Dose Acute Toxicity Testing for Pharmaceuticals, FDA:CDER:
August 1996
6 ASTM F 1904-98: Standard practice for testing the Biological Responses to Particles in vivo
7 Guidance for Industry: Developing Medical Imaging Drug and Biological Products Part 1:
Conducting Safety Assessments, FDA:CDER:June 2004
8 Guidance for Industry: S7A Safety Pharmacology Studies for Human Pharmaceuticals,
FDA:CDER: July 20-01
9 ISO 10993-5, Biological evaluation of medical deivices-part5: tests for in vitro cytotoxicity
10 ASTM F 1903 – 98: Standard Practice for Biological Responses to Particles in vitro

42
 NCI Alliance for
Nanotechnology

Nanotechnologies

 University and Medical Centers Nanotechnology

 Technology Centers Characterization
of Excellence Laboratory
 National Labs NCI
 NCI Technology Clinical Applications
NIST FDA
 Development Programs

Protocols and Data 43

 Questions/Comments

Contact Info:
Scott E. McNeil
(301) 846-6939
ncl@[Link]

44