characterization and safety of nanomedicines: lessons ... presentation ncac fall...
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Characterization and Safety of Nanomedicines: Lessons Learned from the NCL
Scott McNeil
December 16, 2013
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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
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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
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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.
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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
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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
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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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0.4
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% In
jec
ted
do
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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.
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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.
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Attracting Investment in Nanotech
More than $1 billion in potential funding raised by NCL
collaborators. 12
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Role of Nanoparticle
Physicochemical Properties
Physicochemical properties greatly influence nanoparticle biocompatibility.
Dobrovolskaia M., and McNeil S., Nature Nanotechnology 2007, 2(8):469-78. 13
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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.
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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.
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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
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Gold Nanoparticles
20 nm
0
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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
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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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Carbon Nanotubes
TEM
SEM
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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
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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
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Endotoxin
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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
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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.
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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
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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…
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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
0
5
10
15
20
80 100 120 140 160 180 200 220
0
5
10
15
20
25
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.
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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
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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
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Nickel in the Ni-API liposome is most likely responsible for the induction of IL-8.
0
500
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2500
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3500
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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
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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.
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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
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Case Study #3
Residual Impurities
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• 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
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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
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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
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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
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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
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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: [email protected] Ph # 301-846-6939
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Contact Info:
Scott McNeil
(301) 846-6939
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.
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