Characterization and Safety of Nanomedicines: Lessons Learned from the NCL

Scott McNeil

December 16, 2013

Outline

• Characterization Matters!

• Properties to Monitor

• PCC linkage to biocompatibility

• Know What You Have

• Vendor claims vs. data

• Endotoxin

• Case Studies

• Coating Instability

• Biocompatibility of Components

• Residual Impurities

2

NCL Concept of Operations

• The NCL was established in 2004 as an interagency collaboration among

NCI, NIST, and FDA. The lab’s mission is to accelerate the translation of

promising nanotech cancer drugs and diagnostics.

• NCL performs preclinical characterization of nanomaterials, including:

• physicochemical characterization

• in vitro experiments

• in vivo testing for safety and efficacy

90% of NCL’s efforts support the extramural community. 3

NCL is a Translational Resource

• NCL provide independent verification of results can help attract investment.

• Focus on questions related to “translatability”:

• Publication vs. commercialization

• Manufacturing complexity

• Economics (costs to produce, potential for return on investment)

• Quality/regulatory requirements

• Advantage over existing therapies

• Repeat player with FDA: NCL provides submitters a preview of what FDA may be concerned with based on past experience.

G. Naik, Scientists' Elusive Goal: Reproducing Study Results, Wall Street Journal, December 2, 2011

NCL provides independent validation of results, de-risks products.

4

Size/Size Distribution • Dynamic Light Scattering

• Electron Microscopy (TEM, SEM, cryo)

• Atomic Force Microscopy

• Field Flow Fractionation (FFF), SEC-MALLS

Composition • TEM with EDX

• ICP-MS

• Spectroscopy (NMR, CD, Fluorescence, IR, UV-vis)

Purity • Chromatography

• Capillary Electrophoresis

Surface Chemistry • Biacore

• Zeta Potential

Stability • Stability can be measured with any number of instruments with respect to time,

temperature, pH, etc.

CryoTEM

ICP-MS

FFF

AFM

http://ncl.cancer.gov/instrumentation.asp

The NCL Assay Cascade: Physicochemical Characterization

5

The NCL Assay Cascade: In Vitro Cascade

Sterility • Bacterial/Viral/Mycoplasma

• Endotoxin

Cell Uptake/Distribution • Cell Binding/Internalization

• Targeting

Blood Contact Properties

• Plasma Protein Binding

• Hemolysis

• Platelet Aggregation

• Coagulation

• Complement Activation

• Cytokine Induction

Toxicity • Oxidative Stress

• Cytotoxicity

http://ncl.cancer.gov/working_assay-cascade.asp 6

Initial Disposition Study • Tissue Distribution

• Clearance

• Half-life

Immunotoxicity • 28-day screen

• Immunogenicity (repeat dose tox study)

Dose-Range Finding Toxicity • Blood Chemistry

• Hematology

• Histopathology

• Gross Pathology

Efficacy • Therapeutic & Imaging

• Transgenic and xenograft models

http://ncl.cancer.gov/working_assay-cascade.asp

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Time (hours)

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14C

/m

L

3H-Liposome

14C-Ceramide

Plasma Profile

Dual Radiolabels

The NCL Assay Cascade: In Vivo Cascade

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Laboratory Animal Sciences Program

Laboratory of Molecular Technology

Small Animal Imaging Program Protein Chemistry Lab

Electron Microscopy Lab

Laboratory of Proteomics and Analytical Technology

Antibody Characterization Lab

Optical Microscopy and Analysis Lab

Protein Expression Lab

Laboratory of Cell Mediated Immunity

Clinical Support Lab

FNL/NCL Capabilities

Scale-Up Assistance • Batch-to-batch consistency • Process design and optimization • Quality control • Developing methods for in-process

testing

Analysis of Clinical Samples

In Vitro Screening • Blood contact properties • Toxicity • Immune cell functions

Chemistry • Size • Composition • Surface functionality • Compatibility in

biological matrices

In Vivo Screening • ADME-Toxicity • Efficacy • Pharmacokinetics • Drug Metabolism • Immunotoxicity

Reformulation

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Types of Samples

Over 300 Nanomaterials Characterized

Source of Samples • NCL accepts samples from all research

entities.

• NCL has characterized over 300 different nanomaterials and a wide range of various platforms.

• NCL has an average of 15 active collaborations at any given time.

• NCL characterizes an average of 75 samples each year.

9

NCL Collaborators

10

Success Stories: NCL Submissions Now in

Clinical Trials

IND Dec 2009

• ATI-1123 PEGylated nanoliposomal formulation of docetaxel.

• Phase I safety study in patients with advanced solid tumors complete in 2012.

IND 2011

• BIND-014 docetaxel-encapsulated PLGA nanoparticle-aptamer conjugates.

• Binds PSMA expressed on prostate cancer cells.

• Phase I safety study in patients with advanced or metastatic cancer ongoing.

Phase 1 Complete in 2008

• AurImune® PEGylated colloidal gold nanoparticle-TNFa conjugates.

• Phase II study in combination with Taxotere to start in 2013.

IDE 2008

• Silica-core gold-shell particle for photothermal ablation with NIR irradiation.

• Pilot safety study in head and neck cancers ongoing; efficacy study in lung tumors started in 2012.

IND 2010 • PNT2258 liposome-encapsulated

oligonucleotide for breast and lung cancer.

• Phase I safety study in patients with advanced solid tumors ongoing.

IND 2013

• PDS0101, a Versamune® HPV antigen nanoparticle, is cationic (positively charnged), so rapidly taken up by the immune system.

• PDS0101 is a treatment for human papilloma virus (HPV) and cervical cancers.

11

Attracting Investment in Nanotech

More than $1 billion in potential funding raised by NCL

collaborators. 12

Role of Nanoparticle

Physicochemical Properties

Physicochemical properties greatly influence nanoparticle biocompatibility.

Dobrovolskaia M., and McNeil S., Nature Nanotechnology 2007, 2(8):469-78. 13

Re

na

l C

lea

ran

ce

Bil

iary

Cle

ara

nc

e ?

Cytotoxicity

(Surface Reactivity) RES

Recognition

(EPR Effect)

Size

(Rigid Core)

Hyd

rop

ho

bic

ity

Zeta

Po

ten

tial

(+)

(–)

0

Hi

Low

1 nm 220 nm

Nanoparticle Biocompatibility

Cytotoxicity

Dose (mg/mL)

% C

on

tro

l Lessons Learned: Biocompatibility

McNeil (2009), Wiley Interdisciplinary Reviews: Nanomedicine and Nanobiotechnology, 1:264-271.

Nel et al. (2009), Nature Materials 8: 543-557.

Cover of Advanced Drug Delivery Reviews, June, 2009.

Physicochemical parameters contribute to toxicity.

14

30 nm Gold colloids in PBS 30 nm Gold colloids incubated in plasma

33 nm 76 nm

Size in a Biological Context

TEM AFM

DLS

TEM AFM

DLS

27 nm

28 nm

29 nm 31 nm

Dobrovolskaia et al, (2009), Nanomed. Nanotechnol. Biol. Med., 5:106-117.

Multiple orthogonal methods needed to characterize size.

15

Surface Ligand Density

Uncoated

nanoparticles

PEG-coated

nanoparticles

Protein analysis

by 2D PAGE

TEM analysis of particles uptake by

macrophages

2m 2m

Uncoated

nanoparticles

PEG-coated

nanoparticles

PEG-coated

nanoparticles

Protein analysis

by 2D PAGE

Protein analysis

by 2D PAGE

TEM analysis of particles uptake by

macrophages

TEM analysis of particles uptake by

macrophages

2m 2m2m 2m2m2m 2m2m

Dobrovolskaia et al., (2008), Mol.Pharm., 5:487-495.

in vitro

Paciotti J. et al.,(2004), Drug Delivery,11:169-183.

in vivo

Difference in surface characteristics can cause

dramatically different in vivo outcomes.

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Know What You Have

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Formula: CeO2

Purity: 99.5% minimum (based on rare earth oxide impurities)

Formula Weight: 172.12 g/mol

Melting Point: 2600°C

Density: 7.132 g/mL

Form: 15-30 nm average particle size, powder

Cerium Dioxide

Manufacturer-Stated Specs:

What the Material Actually Looks Like:

• Micron-sized aggregates/agglomerates

• Largely insoluble in aqueous media

Vendor used BET for measuring size –

BET is a surface area measurement,

not accurate for size measurements

BET = Brunauer, Emmet, and Teller 18

Gold Nanoparticles

20 nm

0

5

10

15

20

25

30

35

40

45

50

0 5 10 15 20 25 30 35 40

Diameter (nm)

Fre

qu

en

cy

14.0 nm eq. sphere

diameter

Different techniques are sensitive to different size/shape populations.

Different size/shape particles may have different biodistribution and toxicity.

Z-ave PdI

82.1 nm 0.168

TEM

DLS

19

Colloidal Silver Nanoparticles

Advertised as:

However…

unagglomerated, monodisperse, spherical silver nanoparticles

Silver nanoparticles were manufactured using a gold core. Range of sizes and shapes present.

Vendor reported: TEM diameter size distribution, Ag concentration, UV-vis spectral properties

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21

Carbon Nanotubes

TEM

SEM

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90

100

0 500 1000 1500 2000 2500W

idth

(n

m)

Length (nm)

Fraction A

Fraction B

Fraction C

Fraction D

Fraction E

CFFF

CNTs will exist in a variety of sizes, shapes, and

agglomeration states.

Vendor specs: OD 10-20 nm, length 0.5-2 μm

Vendor specs

Reality

Iron Oxide

Three different sources of iron oxide nanoparticles

Iron

Oxide

Shape and size can vary widely.

Z-Avg:

PdI:

55.3 nm

0.058

46.2 nm

0.113

82.9 nm

0.124

22

50 nm 50 nm 50 nm

TEM

DLS

Endotoxin

23

24

What is Endotoxin?

Gram-negative Bacteria

Lipopolysacharide (Endotoxin)

Endotoxin is very common in tap water, air, glassware and

other laboratory reagents and tools.

• Very potent immunostimulant

• Picogram quantities are enough to

induce cytokine storm

• MTD in human 50 EU/kg

• FDA mandated limit for drugs and

medical devices 5 EU/kg

• Exposure may lead to life-threatening

conditions:

- Septic Shock Syndrome (SSS)

- Tissue damage (lungs, kidney,

liver)

- Multiple Organ Failure (MOF)

- Disseminated Intravascular

Coagulation (DIC)

MTD = maximum tolerated dose; EU = endotoxin units; 1 EU = 100 pg

Nanoparticle Interference

Formulations can either suppress or enhance endotoxin detection.

Run all proper controls.

25 LAL = limulus amebocyte lysate

End-point Chromogenic LAL

polymeric NP 1

polymeric NP 2

Dobrovolskaia, M. A., et al. Choice of Method for Endotoxin Detection

Depends on Nanomaterial Type. Nanomedicine (Lond.), 2013, In Press.

Coordination between Agencies

• Physicochemical characterization of various ENMs for a nationally concerted risk/hazard assessment.

Collaboration and strategic planning helped avoid time consuming

setbacks & inconclusive results.

NCL pre-screened all ENMs used in this study.

NCL Particle

Designation Nanomaterial

Endotoxin, Kinetic

Turbidity LAL

First test

lots

New, Large-

scale lots

NIEHS-1 20 nm, citrate

stabilized silver 42.7 EU/mL < 0.5 EU/mL

NIEHS-2 20 nm, PVP

stabilized silver 113 EU/mL ≤ 2.2 EU/mL

NIEHS-3 110 nm, citrate

stabilized silver 0.42 EU/mL < 0.5 EU/mL

NIEHS-4 110 nm, PVP

stabilized silver 144 EU/mL < 0.5 EU/mL

26

Case Study #1

Coating Instability and Agglomeration

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Gold shell

PEG

Core

• Two batches of core shell

nanomaterials appeared identical

to physicochemical

characterization.

• In tox studies, 1st batch caused

extensive lung lesions, 2nd batch

was largely benign.

• What’s causing the dramatically

different safety profiles of

seemingly identical batches?

Core Shell Nanoparticles

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Gold nanoparticle Batch 1

Gold nanoparticle Batch 2

14-day ADME-Tox Study in Rats

Extensive pigmentation in liver, spleen, lungs, ovaries, muzzles. Treatment-related

granulomous lesions in lungs.

Much less pigmentation. Few, statistically insignificant, mild lung lesions.

Dramatic Difference In Vivo

Pyogranulomatous

Inflammation-Lung- H&E-40x

Differences between batches of

nanoparticles were not apparent

by physicochemical

characterization…

29

PCC: No Difference in Size, Zeta Potential

No significant difference between batch

1 and batch 2 in terms of size, charge,

or polydispersity.

Sample

Z-Avg

(nm) PdI

Vol-Peak

(nm) %Vol

Batch 1 165 ± 1 0.114 ± 0.013 176 ± 2 100 ± 0

Batch 2 171 ± 1 0.060 ± 0.022 180 ± 2 100 ± 0

Sample Zeta Potential

(mV)

Batch 1 -7.2 ± 0.5

Batch 2 -8.0 ± 0.7

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80 100 120 140 160 180 200 220

Batch 2: 147 ± 14 nm

Batch 1: 157 ± 16 nm

Diameter, nm

Diameter, nm

TEM DLS

Zeta Potential

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Batch 1 Batch 2

In Vitro Disparity: Batch 1 binds significantly more

plasma proteins than Batch 2.

Protein Binding

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centrifugation

Nanoshell

formulations

(Batch 1 and 2)

Supernatant

Particles pellet

SDS PAGE

Difference in PEG Coatings

PEG

STD Batch1 Batch2 Batch1 Batch2

Supernatant Particles

PEG

Barium Iodine Gel Staining

The PEG was dissociating from the

particles over time, ending up in solution.

This difference in coatings was subtle

enough not to be detected by PCC.

32

Case Study #2

Biocompatibility of Components

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Ni-API Liposomes

NCL in vitro studies indicated

the control (including the nickel

counter ion) liposome was

significantly toxic.

NCL conducted in vivo tox and

efficacy studies to see if this

particle was also toxic in

animals…

Ni-API complex

PEG

34

Water - 10x Water - 100x 10 mM NaCl - 10x 10 mM NaCl- 100x PBS - 10x PBS - 100x

Size Distribution by Intensity Size Distribution by Intensity

Diameter, nm Diameter, nm

Cryo EM

Ni-API Liposome

PCC: Monitoring in Solution

DLS

Ni-only control and Ni-API

liposomes have similar

size by DLS; Cryo EM

reveals different structure.

Single-lamellar structures

Multi-lamellar structure

Ni-only Control Liposome

Ni-only Control

Liposome ~100 nm

Ni-API Liposome

~100 nm

35

Nickel in the Ni-API liposome is most likely responsible for the induction of IL-8.

0

500

1000

1500

2000

2500

3000

3500

4000

4500

NC PC API Liposome Ni-Liposome Ni-Liposome-API

IL-8

, p

g/m

L

Ni-API-Liposome

IL-8 Induction

Literature reports induction of IL-8 gene expression by nickel and found a nickel-responsive element in the promotor region of the IL-8 gene in human alveolar macrophages and epithelial cells: Barchowsky A., et al. A pathway for nickel-induced IL_8 expression. J.Biol. Chem. 2002, 277 (27): 24225-24231 Soucy NV, et al. Nickel induces IL-8 expression in human airway epithelial cells. The Toxicologist 60:317, 2001. 36

15

17.5

20

22.5

25

1 3 5 7 9 11 13 15 17 19 21 23 25 27 29

bo

dy

we

igh

t (g

ms)

measurement days

PBS

NCL 161 (6mg/Kg)NCL 148 (6 mgAs/Kg)Trisenox (6 mgAs/Kg)

PBS

Ni-only Liposome (6mg/kg)

Ni-API Liposome (6mg API/kg)

Free API (6mg API/kg) * *

*

* Significantly different from control P 0.05 (Dunnett test)

In Vivo Study

Ni-only liposome more toxic than free API.

37

Toxicity Associated with Nickel

Fibrinoid

necrosis

Inflammatory

cells

normal

blood

vessel

Serosal blood vessel

Free API (20x) Ni API Liposome 20x

Free API (20x) Ni API Liposome 20x

Casts in

Tubule lumen

Significant nickel-related toxicity with Ni-containing liposomes;

pathology consistent w/ an inflammatory response.

Kidney

38

Case Study #3

Residual Impurities

39

• GNRs prepared using

cetyltrimethylammonium bromide (CTAB), a

cationic surfactant.

• Purified with polystyrenesulfonate (PSS), an

anionic polyelectrolyte often used as a

surrogate peptizing agent, followed by

chloroform extraction and ultrafiltration.

Gold Nanorods

Gold Nanorods

40

DLS and TEM show that the GNRs are relatively

monodisperse. Several lots of GNR are uniform in size and

shape. Only small amounts of square and irregular shaped

“nanorods” (less than 1%) were observed.

Physicochemical Characterization

Irregular Shapes < 1%

DLS Size Distribution by Intensity

Diameter, nm

Smaller peak is an artifact of rotational diffusion anisotropy.

~ 60 nm

Length ~70 nm

Width ~30 nm

41

Published in: Alexei P. Leonov; Jiwen Zheng; Jeffrey D. Clogston; Stephan T. Stern; Anil K. Patri; Alexander Wei; ACS Nano 2008, 2, 2481-2488.

DOI: 10.1021/nn800466c. Copyright © 2008 American Chemical Society

LLC-PK1 cells

HepG2 cells

KB cells

MTT Assay

In Vitro Cell Viability Assay

Implied: GNRs are cytotoxic

Empirical: Gold is benign

42

Retentate/Filtrate Comparison

Stock Retentate Filtrate %

Co

ntr

ol C

ell V

iab

ility

LLC-PK1 MTT Cytotoxicity Assay

filter

GNR

formulations

Filtrate (buffer)

Retentate (GNRs)

Reality:

Cytotoxicity was associated with the buffer, not the GNRs.

Toxic

GNR = gold nanorod; LLC-PK1 = porcine kidney cell line; MTT = 3-(4,5-Dimethylthiazol-2-Yl)-2,5-Diphenyltetrazolium Bromide 43

Summary

• Physicochemical Characterization Matters!

• Physical and chemical properties contribute to a

nanomaterial’s biocompatibility

• Know What You Have

• Manufacturer’s specifications may not always be right

• Perform characterization under relevant conditions

• “Nano” May Not Be To Blame

• Use biocompatible components

• Purify

• Monitor stability

44

How to Apply for NCL Characterization

The NCL has a two-phase application process. For detailed information on submitting a proposal, please visit http://ncl.cancer.gov/working_application-process.asp.

• Brief (2-3 page) White Paper

• Quarterly deadlines (next: Mar 1st)

• Specific questions from review committee

• Part II presentation & discussion with NCL scientists - in person or via webex

• 50% acceptance rate for qualifying applications

• NCL resources are FREE !

Thinking of applying? Have questions?

Email: ncl@mail.nih.gov Ph # 301-846-6939

45

Contact Info:

Scott McNeil

(301) 846-6939

ncl@mail.nih.gov

http://ncl.cancer.gov

Funded by NCI Contracts N01-CO-12400 and HHSN261200800001E.

Pavan Adiseshaiah, Ph.D.

Jennifer Grossman, Ph.D.

Sarah Skoczen, M.S.

Matthew Hansen, M.S.

Timothy M. Potter, B.S.

Jamie Rodriguez, B.S.

Jeffrey D. Clogston, Ph.D.

Rachael M. Crist, Ph.D.

Christopher B. McLeland, B.S., M.B.A.

Barry W. Neun, B.S.

Sonny Man, M.S.

Nanotechnology Characterization Lab

Elizabeth Befekadu, B.S.

Nick Panaro, Ph.D.

Gloryvee Rivera, B.S.

Wendi Custer, B.A.

Diana C. Haines, D.V.M., DACVP

Sarah Anderson, M.S.

Abdullah Mahmud, Ph.D.

Anna Ilinskaya, Ph.D.

Vishakha Ambardekar, Ph.D.

Alpana Dongargaonkar, M.S.

Ulrich Baxa, Ph.D.

Kelly Benauer

Pathology/Histotechnology Lab

Electron Microscopy Lab

Scott E. McNeil, Ph.D.

Anil K. Patri, Ph.D.

Stephan T. Stern, Ph.D., DABT

Marina A. Dobrovolskaia, Ph.D.

Supporting Staff:

Acknowledgements

Becky Schneider, B.S.

46