ROMP based polymer nanoparticles for the targeted delivery of antitumor agents

Cell 2000, 100, 57-70.* American Cancer Society: www.cancer.org Self-sufficiency in growth signals Insensitive to anti-growth signals Evading apoptosis Sustained angiogenesis Tissue inv

ROMP-Based Polymer Nanoparticles

for The Targeted Delivery of

Antitumor Agents

Paul A Bertin, DeeDee Smith, Julianne M Gibbs, Sang-Min Lee, Courtney Phillips, Emily Pentzer, and SonBinh T Nguyen

Department of Chemistry and Center for Nanofabrication and Molecular Self-Assembly Northwestern University, Evanston, IL 60208

Julianne M Gibbs Paul A Bertin DeeDee Smith

Sang-Min Lee

Nanotechnology and Medicine

Chemistry Physics Engineering

Nanotechnology

Bio-Biology Medicine

“Nanotechnology has the potential to make significant contributions to disease

prevention, detection, diagnosis, and treatment.” **

**Srinivas, P R Laboratory Investigation 2002, 82, 657-662.

Hanahan, D.; Weinberg, R A Cell 2000, 100, 57-70.

* American Cancer Society: www.cancer.org

Self-sufficiency

in growth signals

Insensitive to anti-growth signals

Evading apoptosis

Sustained angiogenesis

Tissue invasion &

Metastasis

Infinite replicative potential

Selectivity of existing cytotoxic chemotherapy for malignant cells over

healthy cells is low, resulting in life-threatening side effects.

How can selectivity be improved??

Cancer: An Attractive Target

2005 Projection: 1.37 million new cases in US

alone; 570,000 deaths *

Motivation for Targeted Drug Delivery Research

• Problems associated with small molecule drugs include:

– Poor water solubility – Serious side effects from interaction with non-target cells – Rapidly eliminated from the body

– Drug metabolism decreases activity and increases toxicity

• Research efforts focus on the design of appropriate “bullets” to

address these problems

 Soluble polymers

 Microcapsules

 Liposomes

 Polymeric Micelles and Nanoparticles

Why do we need a “magic bullet”?

1 Core-shell structure 3 Spatial Recognition

Adenovirus Type 2

Virus—A Natural Delivery Vehicle

Polymers as Functional Materials

Organic &

Polymer Chemistry

Biological Chemistry

Advantages of Polymer Therapeutics

• Increased solubility/stability

• Reduced immunogenicity

• Prolonged plasma half-lie through prevention and

avoidance of RES and MPS

• Requires less frequent dosing

“Nanotechnology applied to medicine will bring significant advancement

to the treatment of cancer These hybrid systems can be viewed as the

1st generation of nanomedicines.”

“Using advanced polymer chemistry and precision engineering at the

molecular level, together with an appreciation of the pathophysiology of

normal and disease tissue will help to realize the full therapeutic potential of

the post-genomics era.”

Duncan et al TRENDS in Biotechnology 2006, 24, 39-47.

Passive Targeting Systems

HN O Ph

O NH

N H

O

O HO

O HO

O HO

Doxorubicin (DOX)

DOX

Hydrophobic Hydrophilic

65 nm

combretastatin

Sengupta, S et al Nature 2005, 436, 568-572.

DOX -PLGA PEG-liposome

“Nanocell”

200 nm

PK1

Requirements for Drug Delivery

• Drug delivery particle should self-assemble from drug

molecules, components for targeting, and components

for stability

• Particle size should enable effective penetration in target

tissue and cells (< 400 nm for tumor tissue)

• Drug delivery particles should be stable and biologically

inert before reaching their target

• Drug release should result from interactions with the

target cells

• Components of drug delivery system should easily be

removed from the organism

Polymeric Nanoparticles as Model

Drug Delivery Vehicles

Magic Bullet?

• Core-shell nanostructures with control over morphology, size, and size distribution

• Incorporate multiple therapeutic agents (drug, DNA, protein), imaging agents, and penetrating peptides into a biodegradable or removable (< 45 kDa) platform

• Hydrophilic components should resist protein adsorption and prevent secondary aggregation

• Targeting groups on the surface should allow for site-specific drug delivery

Ferrari, M Nature Rev Cancer 2005, 5, 161-171.

Advantage: multicomponent payload per biorecognition event

• Equilibrium favors polymer formation due to the alleviation of ring

strain

• Highly functional group tolerant, works at mild temperature

• Amenable to the synthesis of monodisperse block copolymers

Ring-Opening Metathesis Polymerization (ROMP)

Ru Cl

Aldrich - 55% endo

X

H X

+

Diels-Alder - >85% endo

OH O

Keith J Watson

A Versatile ROMP Synthon: 100%

exo-Norbornen-2-ol

A Toolbox for Monomer Development

OH

n

O O

OH O

PCy 3

PCy 3

hydrophobic hydrophilic

Block Copolymer Synthesis

Preparation of Polymeric Nanoparticles

Bertin, P A et al

Macromolecules 2004, 37,

8364-8372.

Bertin

In Vitro Drug Release

In vitro release of indomethacin from copolymer

IND35-b-PEO7 nanoparticles after 48 h in pH = 3.0

H2O/DMSO (4:1, v/v) at () 25 C and () 37 C

After 48 h at pH=3.0, 20% of the indomethacin is released.

Bertin, P A.; Watson, K J.; Nguyen, S T Macromolecules 2004, 37, 8364-8372.

pH = 3.0

Dialysis Experiment

Synthesis of Monomers

PEO conjugated norbornene Doxorubicin norbornene(carbamate-linked)

Carbamate is stable at

pH 7.4, and is hydrolyzed at pH 5 and

below.

PEO15 DOX15

-b-1.19 10236

6300 PEO15

PDI

Mw(Daltons)

Mn(Daltons) Polymer

Theoretical Mw of PEO 15 -b-DOX 15 = 17901

Nanoparticle Formation

Particles show narrow size

distribution around 230 nm

In Vitro Release of Doxorubicin

DAPI + DOX DOX

DIC + DOX DIC

63X

still on the cell membrane

Passive Uptake

DAPI + DOX (fixed)

Passive Uptake

Most common cancer in infants with 650 new cases each year

In 7 of 10 cases the disease is not diagnosed until it has metathesized

Common drugs used to treat neuroblastoma:

-bladder inflammation and

kidney damage -hearing loss and kidney damage

-heart damage and skin damage if leakage from administrative vein

Side effects associated with treatment:

Nausea, vomiting, hair loss, mouth sores, depression of immune system, bone marrow suppression

Cancer affecting immature nerve cells, most commonly in the adrenal glands and the sympathetic nervous system ganglia of the abdomen

Medline Plus: www.nlm.nih.gov/medlineplus/neuroblastoma.html

Free Doxorubicin vs Doxorubicin PNP

SKN-SH Wild Type Cells

SKN-SH Wild Cells

SKN-SH Wild Cells with Dox Nanoparticles

With Dr Bernard Mirkin

With Dr Bernard Mirkin

SKN-SHRDOX6 Resistant Cells

SKN-SHRDOX6 Resistant Cells with DOX

Nanoparticles

SKN-SHRDOX6 Resistant Cells

Free Doxorubicin vs Doxorubicin PNP

Cholesterol Control

Cholesterol Control Polymers

21153 1.04

22862 21798

PEO12-b-Chol25

21424 1.02

19509 18980

PEO9-b-Chol28

21096 1.03

22234 21551

PEO 6 -b-Chol 30

16690 1.22

16900 13807

PEO15-b-Chol15

Theoretical MnPDI

Cholesterol Control Nanoparticles

0.01 wt% Nanoparticles

Particles show a narrow size distribution around 240 nm

Indomethacin Control Particles

Particles show a narrow size

distribution around 270 nm

Control Response Curves

With Dr Bernard Mirkin

SKN-SH Wild Type Cells SKN-SH-RDOX6 Resistant Cells

Active Tumor Targeting

• Many cancer cells overexpress receptors related to signaling pathways

for cell growth (HER-2, ανβ3 integrin, folate receptor)

• Ligand-targeted therapeutics allow for selective tumor uptake

Bertin and Davis

O

O Ts 6

R = OTs

Bioconjugation (and/or)

anti-HER-2 IgY (HER-2 overexpressed in 25% of breast cancers)

Bcl-2 antisense oligonucleotide (asODN) (inhibits translation of the antiapoptotic Bcl-2 protein )

D = 170  30 nm Bertin and Davis

DNA-Functionalized Nanoparticles

Gold nanoparticle probes (13 nm) confirmed DNA conjugation and molecular recognition

Thermal Denaturation (at 520 nm)

Bertin and Davis

DNA Control Experiments

Tosylated PNP + GNP probes DNA-PNP + non-complementary GNP probes

Antibody-Functionalized Nanoparticles

Anti-HER-2-PNP + anti-IgY-GNP probes (30 nm)

Control Experiment:

Tosyl-PNP + anti-IgY-GNP probes (30 nm)

Bertin and Davis

Multifunctional Nanoparticle

• Tosylated PNPs functionalized with both antibodies and antisense

oligonucleotides were characterized with GNP probes (hierarchical assembly)

• Novel approach for combining a high-density of chemotherapeutics with

antisense oligonucleotides in a tumor-targeting nanostructure

In vitro Cancer Cell Uptake of PNPs

• Human breast cancer cells (SKBR-3) that overexpress the HER-2

receptor were cultured

• HER-2 receptors are known to internalize immunoliposomes via

endocytosis

Proposed HER-2-mediated endocytotic uptake of PNPs

Nucleus HER-2 receptor

Park, J W et al PNAS 1995, 92, 1327-1331.

Suggests anti-HER-2 IgY mediates PNP uptake

single strand DNA – 16h

DNA-PNP – 16h

Bertin and Davis with Clifton Shen, Bill Russin

incubated with SKBR3 cells

Bertin and Davis with Clifton Shen, Bill Russin

N N

N N

O O

O

O

O O

NH2OH

Gd

Incubate 24 h at 37oC

Outlook: [Gd]-Functionalized PNPs

Smith, with Meade’s group

TEM image of nanoparticles from

IND35-b-PEO[Gd]7 NOT stained).

CONTIN analysis showing the intensity-weighted hydrodynamic diameter distribution of nanoparticles

from IND35-b-PEOTs7 as measured by dynamic light

scattering.

Diagnostics

+

Therapeutics

Outlook: Multiple Drugs Incorporation

O O

N Cl

Cl

PCy 3

PCy 3 CHPh

Outlook: Selective Release

• Incorporation of enzyme-specific, tetrapeptide

Donde Anderson

Outlook: Polymer-Coated Liposomes for

n 1.

Proposed strategy for stabilizing arsenic-containing liposomes as

shell-crosslinked polymeric nanoparticles.

Sang-Min Lee, with O’Halloran group

• Drug-containing polymeric nanoparticles with surface-reactive groups can be

conjugated with antisense oligonucleotides and antibodies for targeted delivery of multi-component therapeutic regimes

• Multifunctional PNPs were internalized in breast cancer cells

Doxorubicin-containing PNPs shown enhanced activity against drug-resistant neuroblastoma cells

Clifton Shen

Steve Rosen Nancy Krett Thomas O’Halloran

Haimei Chen

ARCS Foundation

Malkin Scholarship

Thank you for your attention!

Questions?