Transcript Unither Conference Talk April 1

James Leary, Ph.D.
Professor of Nanomedicine
Purdue University & Member of
Birck Nanotechnology Center
1st Annual Unither Nanomedical & Telemedical Technology Conference
Hotel Manoir Des Sables, Orford (Quebec)
April 1- 4, 2008
Programmable Nanoparticles for
Drug/Gene Delivery in Regenerative
Nanomedicine
James F. Leary, Ph.D.
SVM Endowed Professor of Nanomedicine
Professor of Basic Medical Sciences and
Biomedical Engineering
Member: Purdue Cancer Center; Oncological Sciences Center;
Bindley Biosciences Center; Birck Nanotechnology Center
Email: [email protected]
The Progression of Medicine
Conventional
“Modern”
Medicine
“Personalized” or
“Molecular”
Medicine
+
Nanomedicine
Single-cell
Medicine
From an Early Era of Nanomedicine …
NASA Press Release: Today, April 13, 2000, NASA Administrator Daniel S.
Goldin and National Cancer Institute (NCI) Director Dr. Richard Klausner
signed a Memorandum of Understanding to develop new biomedical
technologies that can detect, diagnose and treat disease here on Earth and in
space. The development of such technologies will improve life on Earth and
one day revolutionize medicine and space travel.
UTMB: As one of the original 13 groups (7 NASA-funded, 6 NCI funded)
nationwide funded by this program, UTMB and collaborating scientists are
developing nanomedical systems for NASA to continuously repair and combat the
effects of radiation on astronauts. (This research was funded by the Biomolecular,
Physics and Chemistry Program under NASA-Ames grant NAS-02059 (BAA N01CO-17014-32 )
http://science.nasa.gov/headlines/y2002/15jan_nano.htm
January 15, 2002: It's like a scene from
the movie "Fantastic Voyage." A tiny
vessel -- far smaller than a human cell -tumbles through a patient's bloodstream,
hunting down diseased cells and
penetrating their membranes to deliver
precise doses of medicines.
Only this isn't Hollywood. This is real
science.
Right: Tiny capsules much smaller than
these blood cells may someday be injected
into people's bloodstreams to treat conditions ranging from cancer to radiation damage. Copyright
1999, Daniel Higgins, University of Illinois at Chicago.
http://www.nanohub.org/courses/nanomedicine
MOLECULAR CYTOMETRY FACILITY - 2008
BIONANOTECHNOLOGY
BIO-INSTRUMENTATION
 Engineering Nanomedical
Systems1,2,3,5,8, +
 Nanostructure characterization
( XPS, AFM, TEM)2,3,5,8, +
 High-throughput cytometry1,2,6, 7,+
 Microfluidic cytometer/sorter1,2,3,7, +
 LEAP interactive molecular
imaging/sorting/opto-injection1,6,9, +
 Nanomaterials/chemistry2,3,5
 In-vitro/In-vivo molecular imaging
+
2,5,6,9
(optical, MRI, thermal)1,2,5,8,10, +
 Biomolecular sensors
Peptide, aptamer,
CYTOMICS
 Magnetic sorting1,2,3,5, +
gene synthesis,
 Circulating cancer cells
screening1,2,3,5,6,9, + (breast & prostate cancer,
 SPR1,2,3,4,7, +
cancer stem cells)1,2,5,6,8 +
Faculty & Staff
1= Leary (Director)
2= Reece
3= Cooper
+ = Collaborators
 Detection of pathogens1,2,3,4,7,+
 Regenerative medicine
(gene expression & silencing)1,2,6,9
 Stem/progenitor cell isolation
& characterization1,2,6,9, +
 Animal studies2,5,8, +
Graduate Students
4= Seale-Goldsmith
5= Zordan
6= Grafton
7= Haglund
8= Eustaquio
 Existing areas
 New areas
January, 2008
9= Key
Use of Ultra-High Speed Flow Cytometry and Cell Sorting to Select
Targeting Aptamers and to Evaluate Targeting to Rare Cells
This high-speed flow cytometer/cell sorter is the world’s fastest
instrument and is used for separating rare cells or particles of interest.
Sorting of thioaptamer combinatorial chemistry library beads with bound
protein, is one way to isolate a specific drug. Up to 100 million drug candidates
can be screened in a single day using high-throughput technologies.
A high-speed (>10,000 cells/sec), portable (PDAsized), commercially manufacturable, multi-stage
BioMEMS microfluidic cell sorter.
Leary, J.F. "Ultra High Speed Cell Sorting" Cytometry Part A
67A:76–85 (2005)
The Multi-Step Targeting Process in
Nanomedical Systems
(1) Multilayered nanoparticle
(2) Multilayered nanoparticle
targeting to cell membrane
receptor and entering cell
(3) Intracellular targeting to
specific organelle
(4) Delivery of therapeutic
gene
Targeted cell
Targeted cellular organelle
Breast Cancer Cell Targeting with
Peptides from Peptide Libraries
• SKBR3 Cells uptake pf
fluorescent labeled
peptide
• Amino acid sequence LTVSPWY
• Possible targeting peptide
for nanoparticles
Shadidi, M., Sioud, M., Identification of novel carrier
peptides for the specific delivery of therapeutics into
cancer cells, FASEB Journal 2002, 16, 256.
Rapid Prototyping of PeptideGuided Nanomedical Systems
with Quantum Dot Cores
Quantum Dot Nanoparticles for Ex-Vivo
Diagnostics and Rapid Prototyping
Biocoating to make
hydrophilic and
biocompatible (e.g.
PEG)
ZnS cap
Semi-conductor
core material
(e.g. CdSe)
Targeting/entry
promoting molecule
(e.g. a peptide)
Transmission electron
microscopy (TEM) image of
amino-functionalized Qdots.
Size was determined to be
~10 nm.
Receptor-mediated Endocytosis
• Targeting peptide – LTVSPWY
• SkBr3 Breast cancer cell
• Conjugation of targeting peptide
OH
OH
OH
C
OH
O
HO
C
HO
HO
CH
CH
C
H 2N
CH
N
H
O
CH
H
N
O
C
CH
N
H
C
CH
O
O
CH
O
NH
C
N
O
C
C
CH
N
H
C
H2
NH
Fmoc-Cl, 10% Na2CO3
Dioxane
HO
CH
C
FmocHN
H
N
N
H
C
CH
N
H
C
N
C
CH
O
O
NH
O
C
C
CH
N
H
NH
C
H2
CH3
H3 C
CH2
CH3
H3 C
H3 C
CH2
H3C
CH
O
CH
CH
O
CH2
CH
CH2
O
CH3
CH2
O
CH3
CH2
CH
CH3
CH
CH3
LTVSPWY
Fmoc-LTVSPWY +
Quantum Dot
(Shadidi, 2003)
Confirmed by MALDI-MS
NH
NH
2
2
DIEA, TBTU,
HOBt hydrate,
NMP/Water
LTVSPWY
LTVSPWY
Quantum Dot – Peptide conjugated
Peptide targeted Qdot nanoparticles
Drawing is not to scale!
SKBR3 Cell
LTVSPWY
LTVSPWY
QD
14 nm
15 microns
=15,000 nm
The Qdot nanoparticle with PEG layer is
approximately 1/1000th the diameter of
the cell or approximately one billionth
the volume of the SKBr3 human breast
cancer cell.
Biomolecular Targeting: Peptide
• Use of biomolecules offers advantages toward other uptake
mechanisms: Cell receptor is targeted and functions normally
• Peptide offers ease of synthesis and well understood
chemistry.
These are also on the size order of the
nanoparticles.
– QTracker® Cell Labeling Kit (Invitrogen Corporation,
Carlsbad, CA) offers Qdot nanoparticles conjugated to a
universal peptide. This will enter all cell lines.
– Specific peptides will enter only certain cell types; the
focus of nanomedical approach to disease
UNIVERSAL
QDOT
QDOT
PEPTIDE
LTVSPWY
ALL CELL
TYPES
SKBR3 CELLS
ONLY
Confocal Imaging of Qdots with
SKBr3 Cells
• Successful targeting
SkBr3 breast cancer
cell
– Targeting
– Entry
• Did not efficiently
target MCF-7 breast
cancer cells
• Future experiments
– Scrambled peptide
– Mixed cell populations
Peptide conjugated quantum dot
SkBr3 Breast Cancer Cells
MCF-7
CONTROL
SkBr3
EXPERIMENTAL
UNIVERSAL
QDOT
PEPTIDE
ALL CELL
TYPES
MCF-7
CONTROL
SkBr3
EXPERIMENTAL
QDOT
LTVSPWY
SKBR3 CELLS
ONLY
Cytotoxicity: Results
• Fluorescent imaging
– There was distinct indication of changes in cellular
morphology and decrease in Qdot brightness
– The application of UV light to the cells with and without
Qdots did not afford any detection of apoptotic cells as
detected by Annexin V assays of early apoptosis.
(a) Control cells, no nanoparticles
(b) Positive control cells, induced
with hydrogen peroxide
(c) MCF-7 cells with QTracker®
(d) MCF-7 cells with QTracker®
and UV light application
Cytotoxicity: Results
• Confocal imaging
– ROS are present normally in cells. Heightened presence
indicates a state of cellular stress.
– Detection of ROS was observed in the positive control sample
and the QTracker® sample.
Dihydroethidium is shown in red QTracker® is shown in green.
(a) Control
(b) H2O2
(c) QTracker®
Some –in-vivo biodistribution studies
In-vivo peptide targeting of Qdot nanoparticles to
human SKBr3 breast cancer cells in nude mice
In vivo SkBr3 Tumor Study:
Results
a
b
c
Fluorescent microscopy images of in vivo tumor tissue.
(a) Image of control kidney tissue, this sample did not
receive any Qdots.
(b) Image of tumor tissue from a peritumoral injection.
(c) Image of tumor tissue from a tail vein injection.
Qdot Agglomeration
Single Qdot
a
Agglomerated
Qdots
b
(a) In vivo tumor image. (b) Graphic representation of agglomerated Qdots.
NANOPARTICLE AGGLOMERATION:
~1000 - 2000 nm IN DIAMETER
APPROXIMATE: 50 – 100 NANOPARTICLES PER CLUSTER IN DIAMETER
CONSIDERING THREE DIMENSIONS, THE NUMBER OF NANOPARTICLES
PRESENT COULD BE BETWEEN 125,000 AND 106
Nanomedicine – The Future
Biomimicry – Can Nature Provide Some
of the Answers?
Viruses know how to perform a
multi-step targeted process to
infect cells, use the host cell
machinery to produce gene
products, and make copies of
themselves. What if we could
make a synthetic “good virus” that
could deliver therapeutic gene
templates to specific cells, and use
the host cell machinery to produce
therapeutic genes to perform
regenerative medicine in a cell and
cure disease at the single cell level
(and NOT make copies of
themselves!) ?
The challenge of precise drug delivery
and dosage per cell
It is impossible to control the number of nanoparticles
that will bind and be active in a given cell. For
regenerative nanomedicine the drug/gene needs to be
created in-situ and controlled in feedback loops. This is
possible to do with biomolecular sensors controlling
down-stream transient gene therapy inside living cells.
Designing “Programmable” Multifunctional
Nanomedical Systems with Feedback Control of
Gene/Drug Delivery within Single Cells
Cell targeting and entry
Y
Intracellular targeting
Therapeutic genes
Y
Magnetic or Qdot core
(for MRI or optical
imaging)
Biomolecular sensors
(for error-checking
and/or gene switch)
Targeting molecules (e.g. an
antibody, an DNA, RNA or peptide
sequence, a ligand, an aptamer) in
proper combinations for more
precise nanoparticle delivery
Y
Leary and Prow, PCT (USA and Europe) Patent pending 2005
Dealing with the dosing problem: Concept of
nanoparticle-based “nanofactories” –feedback-controlled
manufacturing of therapeutic genes inside living cells for
single cell treatments using engineered nanosystems
Multilayered targeted nanosystem
Y
Y
NF
cell
cell membrane
cytoplasm
nucleus
Therapeutic gene/drug
Gene
manufacturing
machinery
Molecular Biosensor
NF
control switch
The nanoparticle delivery system delivers the therapeutic gene template which
uses the host cell machinery and local materials to manufacture therapeutic
gene sequences that are expressed under biosensor-controlled delivery.
Y
Dealing with the dosing problem: Concept of
nanoparticle-based “nanofactories” –feedback-controlled
manufacturing of therapeutic genes inside living cells for
single cell treatments using engineered nanosystems
Y
Multilayered targeted nanosystem
MNP
cell
cell membrane
cytoplasm
nucleus
Therapeutic gene/drug
Molecular Biosensor
control switch
Gene manufacturing
machinery
Feedback control
Specific molecules inside living
diseased cell being treated
with manufactured genes
Example of multilayered magnetic
nanoparticle for in-vivo use
Prow, T.W., Grebe, R., Merges, C., Smith, J.N., McLeod, D.S., Leary, J.F.,
Gerard A. Lutty, G.A. "Novel therapeutic gene regulation by genetic biosensor
tethered to magnetic nanoparticles for the detection and treatment of
retinopathy of prematurity" Molecular Vision 12: 616-625, 2006
Efficient Gene Transfer with DNA
Tethered Magnetic Nanoparticles
PCR product bioconjugated to magnetic nanoparticle
CMV
EGFP
pA
+
Lipid coated magnetic
nanoparticles tethered with DNA
SPIO
Lipid
Magnetic nanoparticle
tethered with DNA
+
SPIO
Add to cell culture
SPIO
`
Tethered Gene Expression on Magnetic
Nanoparticles for Nanomedicine
1. Prow, T.W., Smith, J.N., Grebe, R., Salazar, J.H., Wang, N., Kotov, N., Lutty, G., Leary, J.F. "Construction, Gene Delivery, and Expression of
DNA Tethered Nanoparticles" Molecular Vision 12: 606-615, 2006a.
2. Prow, T.W., Grebe, R., Merges, C., Smith, J.N., McLeod, D.S., Leary, J.F., Gerard A. Lutty, G.A. "Novel therapeutic gene regulation by genetic
biosensor tethered to magnetic nanoparticles for the detection and treatment of retinopathy of prematurity" Molecular Vision 12: 616-625, 2006b.
http://www.nanohub.org/resource_files/2007/10/03388/2007.09.14-choi-kist.pdf
Our MCF Team and Current Collaborators
Nanochemistry
Don Bergstrom (Purdue)
Combinatorial chemistry/
Drug Discovery
David Gorenstein (UTMB)
Xianbin Yang (UTMB)
Andy Ellington (UT-Austin)
Nanoparticle technology
Nick Kotov (Univ. Michigan)
Kinam Park (Purdue)
Alex Wei (Purdue)
Nanotoxicity studies
Debbie Knapp (Purdue)
James Klaunig (IU-SOM)
MRI Imaging
Tom Talavage (Purdue)
Charles Bouman (Purdue)
Image/confocal/SPR
Paul Robinson (Purdue)
Joseph Irudayaraj (Purdue)
Funding from NIH, NASA,
and Army Breast Cancer
Program
Molecular Cytometry Facility
Director: James Leary
Lisa Reece (SVM) – flow cytometry/
BioMEMS; tissue culture
Christy Cooper (SVM) - bioanalytical
chemistry, nanochemistry, XPS, AFM
Meggie Grafton (BME) - BioMEMS
Emily Haglund (BME) – multilayered
Qdots for ex-vivo nanomedicine
Mary-Margaret Seale-Goldsmith
(BME) – multi-layered magnetic
nanomedical systems
Michael Zordan (BME) – prostate
cancer, rare cell flow/image cytometry
Trisha Eustaquio (BME) – gene
silencing/therapy; interactive imaging
Jaehong Key (BME)- 3D/MRI
imaging
X-ray Photon Spectroscopy
Dmitry Zemlyanov (Purdue)
High-Energy TEM
Eric Stach (Purdue)
Dmitri Zakharov (Purdue)
Atomic Force Microscopy
Helen McNally (Purdue)
Systems Biology
Doraiswami Ramkrishna (Purdue)
Ann Rundell (Purdue)
Robert Hannemann (Purdue)
Magnetic Cell Sorting
Paul Todd (SHOT, Inc)
LEAP Interactive Imaging
Fred Koller (Cyntellect, Inc.)
BioMEMS/Microfluidics
Nanomedicine studies
Debbie Knapp (Purdue)
Deepika Dhawan (Purdue)
Sophie Lelievre (Purdue)
Gerald Lutty (Johns Hopkins U)
Tarl Prow (U. Brisbane, Australia)
Rashid Bashir (Purdue)
Cagri Savran (Purdue)
Kinam Park (Purdue)
Pedro Irazoqui (Purdue)
Huw Summers (Cardiff Univ, UK)
A Few Relevant Recent References
1. Prow, TW, Salazar, JH, Rose, WA, Smith, JN, Reece, LM, Fontenot, AA, Wang, N, Lloyd,
RS, Leary, JF: "Nanomedicine – nanoparticles, molecular biosensors and targeted
gene/drug delivery for combined single-cell diagnostics and therapeutics" Proc. SPIE 5318:
1-11, 2004.
2. Prow, TW, Kotov, NA, Lvov, YM, Rijnbrand, R, Leary, JF: “Nanoparticles, Molecular
Biosensors, and Multispectral Confocal Microscopy” Journal of Molecular Histology, Vol. 35,
No.6, pp. 555-564, 2004.
3. Prow, TW, Rose, WA, Wang, N, Reece, LM, Lvov, Y, Leary, JF: "Biosensor-Controlled Gene
Therapy/Drug Delivery with Nanoparticles for Nanomedicine" Proc. of SPIE 5692: 199 –
208, 2005.
4. Prow, TW, Grebe, R, Merges, C, Smith, JN, McLeod, DS, Leary, JF, Lutty, GA: "Novel
therapeutic gene regulation by genetic biosensor tethered to magnetic nanoparticles for the
detection and treatment of retinopathy of prematurity" Molecular Vision 12: 616-625, 2006
5. Prow, TW, Smith, JN, Grebe, R, Salazar, JH, Wang, N, Kotov, N, Lutty, G, Leary, JF:
"Construction, Gene Delivery, and Expression of DNA Tethered Nanoparticles" Molecular
Vision 12: 606-615, 2006
6.
Seale, M., Haglund, E., Cooper, C.L., Reece, L.M., Leary, J.F. "Design of programmable
multilayered nanoparticles with in situ manufacture of therapeutic genes for nanomedicine"
Proc. SPIE 6430: 643003-1-7, 2007.
7.
Seale, M., Zemlyanov, D., Cooper, C.L., Haglund, E., Prow, T.W., Reece, L.M., Leary,
J.F. “Multifunctional nanoparticles for drug/gene delivery in nanomedicine” Proc. SPIE 6447:
64470E-1-9, 2007.
8. Leary, J.F. and Prow, T.W. Multilayered Nanomedicine Delivery System and Method
PCT/US05/06692 on 3/4/2005.