James Leary, Ph.D.Professor of Nanomedicine

Purdue University & Member of

Birck Nanotechnology Center

Programmable Nanoparticles for Drug/Gene Delivery in Regenerative

Nanomedicine

James F. Leary, Ph.D.SVM Endowed Professor of NanomedicineProfessor of Basic Medical Sciences and

Biomedical EngineeringMember: Purdue Cancer Center; Oncological Sciences Center;

Bindley Biosciences Center; Birck Nanotechnology CenterEmail: jfleary@purdue.edu

1st Annual Unither Nanomedical & Telemedical Technology ConferenceHotel Manoir Des Sables, Orford (Quebec)

April 1- 4, 2008

Conventional “Modern”Medicine

“Personalized” or “Molecular” Medicine

NanomedicineSingle-cell Medicine

The Progression of Medicine

+

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.

From an Early Era of Nanomedicine …

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 N01-CO-17014-32 )

Voyage of the Nano-Surgeons

NASA-funded scientists are crafting microscopic vessels that can venture into the

human body and repair problems – one cell at a time.

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://science.nasa.gov/headlines/y2002/15jan_nano.htm

http://www.nanohub.org/courses/nanomedicine

BIO-INSTRUMENTATION

CYTOMICS

BIONANOTECHNOLOGY

MOLECULAR CYTOMETRY FACILITY - 2008

Engineering Nanomedical Systems1,2,3,5,8, +

Microfluidic cytometer/sorter1,2,3,7, +

High-throughput cytometry1,2,6, 7,+

LEAP interactive molecular imaging/sorting/opto-injection1,6,9, +

Regenerative medicine(gene expression & silencing)1,2,6,9

Nanomaterials/chemistry2,3,5 +

Magnetic sorting1,2,3,5, +

SPR1,2,3,4,7, + Circulating cancer cells(breast & prostate cancer, cancer stem cells)1,2,5,6,8 +

In-vitro/In-vivo molecular imaging (optical, MRI, thermal)1,2,5,8,10, + Biomolecular sensors2,5,6,9

Stem/progenitor cell isolation & characterization1,2,6,9, +

Nanostructure characterization( XPS, AFM, TEM)2,3,5,8, +

Faculty & Staff

1= Leary (Director)

2= Reece

3= Cooper

+ = Collaborators

Graduate Students

4= Seale-Goldsmith

5= Zordan

6= Grafton

7= Haglund

8= Eustaquio

9= Key

Peptide, aptamer, gene synthesis,

screening1,2,3,5,6,9, +

Animal studies2,5,8, +

Existing areas New areas

Detection of pathogens1,2,3,4,7,+

January, 2008

This high-speed flow cytometer/cell sorter is the world’s fastest instrument and is used for separating rare cells or particles of interest.

Use of Ultra-High Speed Flow Cytometry and Cell Sorting to Select Targeting Aptamers and to Evaluate Targeting to Rare Cells

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 (PDA-sized), commercially manufacturable, multi-stage BioMEMS microfluidic cell sorter.

Leary, J.F. "Ultra High Speed Cell Sorting" Cytometry Part A 67A:76–85 (2005)

Targeted cellular organelleTargeted cell

Cell membrane

(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

The Multi-Step Targeting Process in Nanomedical Systems

(1) Multilayered nanoparticle

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 Peptide-Guided Nanomedical Systems

with Quantum Dot Cores

Quantum Dot Nanoparticles for Ex-Vivo Diagnostics and Rapid Prototyping

ZnS cap

Biocoating to make hydrophilic and biocompatible (e.g. PEG)

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

H 2 NCH

C

CH 2

O

CHH3 C

CH3

NH

CHC

CH

O

HO

CH 3

HN

CH

C

CH

O

CH 3

H3 C

NH

CHC

CH 2

O

HO

N C

O

NH

CH

C

CH 2

ONH

NH

CH

C CH 2

O

OH

O H

LTVSPWYConfirmed by MALDI-MS

Quantum Dot

Fm ocHNCH

C

CH 2

O

CHH 3 C

CH 3

NH

CHC

CH

O

HO

CH 3

HN

CH

C

CH

O

CH 3

H 3 C

NH

CHC

CH 2

O

HO

N C

O

NH

CH

C

CH 2

ONH

NH

CH

C CH 2

O

OH

OH

Fmoc-LTVSPWY + LTVSPWY

Fmoc-Cl, 10% Na2CO3

Dioxane

DIEA, TBTU, HOBt hydrate,

NMP/Water

Quantum Dot – Peptide conjugated

NH2NH2 LTVSPWY

(Shadidi, 2003)

LTVSPWY QDSKBR3 Cell

Peptide targeted Qdot nanoparticles

Drawing is not to scale!

15 microns

=15,000 nm

14 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

PEPTIDE

ALL CELL

TYPES

LTVSPWYSKBR3 CELLS

ONLY

QDOT

QDOT

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 dotSkBr3 Breast Cancer Cells

UNIVERSAL

PEPTIDE

ALL CELL

TYPESQDOT

MCF-7

CONTROL

SkBr3

EXPERIMENTAL

LTVSPWYSKBR3 CELLS

ONLYQDOT

MCF-7

CONTROL

SkBr3

EXPERIMENTAL

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

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.

a b c

Qdot Agglomeration

Single Qdot Agglomerated Qdots

ba

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

(a) In vivo tumor image. (b) Graphic representation of agglomerated Qdots.

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!) ?

Biomimicry – Can Nature Provide Some Biomimicry – Can Nature Provide Some of the Answers?of the Answers?

Nanomedicine – The Future

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.

YY

Y

Y

YY

YYYYY

YY

YY

YY

YY

Y

YY Y Y Cell targeting and entry

Intracellular targeting

Therapeutic genes

Magnetic or Qdot core(for MRI or optical imaging)

Designing “Programmable” Multifunctional Nanomedical Systems with Feedback Control of

Gene/Drug Delivery within Single Cells

Y

YY

YY

YYY

YY

YY

YY Y

Targeting molecules (e.g. an antibody, an DNA, RNA or peptide sequence, a ligand, an aptamer) in proper combinations for more precise nanoparticle delivery

Biomolecular sensors(for error-checking and/or gene switch)

Leary and Prow, PCT (USA and Europe) Patent pending 2005

cell membrane

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.

nucleus

cell

cytoplasm

Therapeutic gene/drug

Molecular Biosensor control switch

YYYYYYYYYY

YYY

YY

NF

NF

Gene manufacturing machinery

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

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

cell membrane

Multilayered targeted nanosystem

nucleus

cell

cytoplasm

YYYYYYYYYY

YYY

YY

MNP

Molecular Biosensor control switch

Gene manufacturing machinery

Therapeutic gene/drug

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

+CMV EGFP pASPIO

PCR product bioconjugated to magnetic nanoparticle

SPIO

Magnetic nanoparticle tethered with DNA

+Lipid

Lipid coated magnetic nanoparticles tethered with DNA

SPIO

`

Add to cell culture

SPIOSPIO

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 CollaboratorsMolecular 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

Combinatorial chemistry/ Drug DiscoveryDavid Gorenstein (UTMB)Xianbin Yang (UTMB)Andy Ellington (UT-Austin)

MRI ImagingTom Talavage (Purdue)Charles Bouman (Purdue)

Nanoparticle technologyNick Kotov (Univ. Michigan)Kinam Park (Purdue)Alex Wei (Purdue)

BioMEMS/MicrofluidicsRashid Bashir (Purdue)Cagri Savran (Purdue)Kinam Park (Purdue)Pedro Irazoqui (Purdue)Huw Summers (Cardiff Univ, UK)

LEAP Interactive ImagingFred Koller (Cyntellect, Inc.)

NanochemistryDon Bergstrom (Purdue)

Nanomedicine studiesDebbie Knapp (Purdue)Deepika Dhawan (Purdue)Sophie Lelievre (Purdue)Gerald Lutty (Johns Hopkins U)Tarl Prow (U. Brisbane, Australia)

Image/confocal/SPRPaul Robinson (Purdue)Joseph Irudayaraj (Purdue)

X-ray Photon SpectroscopyDmitry Zemlyanov (Purdue)

Systems BiologyDoraiswami Ramkrishna (Purdue)Ann Rundell (Purdue)Robert Hannemann (Purdue)

Nanotoxicity studiesDebbie Knapp (Purdue)James Klaunig (IU-SOM)

High-Energy TEMEric Stach (Purdue)Dmitri Zakharov (Purdue)

Atomic Force MicroscopyHelen McNally (Purdue)

Magnetic Cell SortingPaul Todd (SHOT, Inc)

Funding from NIH, NASA, and Army Breast Cancer

Program

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.

A Few Relevant Recent References