Nanotek & Expo 2014

Biodegradable nanobrushes for drug delivery

Eggehard Holler, Hui Ding, Ramachandran Murali, Julia Y. Ljubimova

Cedars-Sinai Medical Center, Los Angeles, USA

Drug

Targeting Agent

Imaging Agent

Linker

Dendrimer Carbon Nanotube

Micelle

Gold Nanoparticle

Third generation ofNano-based Carriers

For Disease Detectionand Therapy

NanoparticleLiposome

Polymer- Conjugate

Synthesis and degradation

FromGlucose

OHHOOCn = 500 to 3000

Physarum:

HOOC OHn = 40 to 80

Aureobasidium:

Poly(β-L-malic acid)

Depolymerisation:Spontaneous& Enzymatic

MalatePurification

Plastic bags Medical support

Drug deliveryMalignancy

Infection

Contrast agentsImaging

Nano scale

αβ

Antisense Oligonucleotide

Monoclonal Antibody (mAb)

Disulfide, cleaved byGlutathion in the

Cytoplasm

Polymalic acid-trileucine copolymer

Prodrug

Targeting

O CH1

C H22

CO

OH

C3

O

O4

CH5

C H2

CO

NH

C OH

O n

Leu-Leu-LeuCOOH

Polymalic acid-trileucine copolymer

Polycefin – Functional Core

HO-CH-CH2-C-OH

O=C

OH=

O

L-Malic acid

Anti-EFGR mAb(Cetuximab)

Anti-mouse TfR mAb

PEG

Antisense RNAEGFR

Cancer cell

Late Endosome

Early EndosomeGluta-thione

Nucleus

Akt

Antisense

EGFR mRNA

EGFR signalling

Endocytosis

EGFR

Active via receptor Extravasation

Mouse TfR

I.V. Injection

Blood vessel

PO4

EPR -effect passive

Preparation of nanodrugs for brain tumor treatment

GBM: Chain-α4 Chain-β1 ( - ) anti-MsTfRmAb anti-HuTfRmAb HER2-positive breast: HER2 ( - ) ( - ) (anti-MsTfRmAb) Herceptin

Triple-negative breast: EGFR ( - ) ( - ) (anti-Ms-TfRmAb) Cetuximab

O O

O OO O OOH OH OH

O O O O O O O O

O O O O O O OO O O O O O O ONH NH NH NH NH NH NH OH

O

CH3

O

SSS S S

OO NH

SS S S

OHN OHN OHN PEG3400

O O

O O

PEG3400

O O

O O

S S

CH3CH3

O O O O

O O OO O O ONH NH NH OH

O

CH3

O

SHCH3

CH3

O

O O

Step-2

Step-1

mAb-1 mAb-2

AON-1

AON-2 AON-3

Polymalic acid

mPEG5000

mPEG5000

Alx-680optional

Primary Triple Negative Breast Cancer

Primary HER2-Positive Breast Cancer

Primary breast cancer Brain metastases

47% survival rate improvementMetastatic HER2-Positive Breast Cancer

Surv

ival

%

0 25 50 75 1000

50

100

79% survival rate improvement

Surv

ival

%

0 10 20 30 40 50 60 70 800

50

100

Days

Metastatic Triple Negative Breast Cancer

Examples for drug delivery by Polycefin variants

Replacement of antibody by affinity peptide: Pro and Contra

Pro Contra

Multiple peptides per conjugate

Less affinity to bind to target

Robust structure Absence of Fc and biological activity

Small size for slender shape of polymeric nanoconjugate

Less passive tissue targeting (EPR)

Increased diffusibilityDeep tissue penetration

Low Stability/reduced longevity in plasma

Reduced immunogenicityHumanization not required

Possibility of unscheduled side reactions

Possibility of multivalency Tendency for aggregation

Chemical fabrication

Easy packaging and delivery.Decreased overall MW of nanodrug and less injectable drug volumen

AngiopepMichel Demeule, et al. (2008) J Parm Exp Therapeut

Target on BBB endothelial cells: LRP-1Guangqing X and Liang-Shang G (2013) Int J Cell Biol

Targeting Brain tumor

1. Example

For Uptake and Imaging: P/PEG2000-Angiopep(2%)/AlexaFluor 680 (0.5%)

Glioblastomacell

Late Endosome

Early Endosome

Nucleus

Extravasation through BBB into glioblastoma

I.V. Injection

Blood vessel

Angiopep

PEG2000

P =

poly

mal

ic a

cid

AlexaFluore 860

Endocytosis

LRP-1LRP-1

PMLSA-Angiopep-2: P/PEG2000-Angiopep(2%)/AlexaFluor 680

Fluorescence Imaging of Glioblastoma-Nude Mouse Model

Tumor

Specific problem with affinity peptides:

Self-association and aggregation because of: Electrostatic complementation

Lipophilic amino acids

OH OHNH

O CH3

CH3

NH

S

NH NH

S

S

AON

R

LLLLLL

OH

NH

O CH3

CH3NH

O CH3

CH3OH

NH

O CH3

CH3N

C

O

OOC

O

OO C

O

OOC

O

OOC

O

OOC

O

OOC

O

OOC

O

OO C

O

OOC

O

OO

PEG3400

NO O

S

NO O

AlexaFluoR 860

H

O CH3

CH3NH

O CH3

CH3OH

OHOH

OHOH2

Nanoconjugate250-500 kDa

(40%) (40%)

(2%) (0.5%)(2%)

Target: HER2Ramachandran Murali et al. (2001) J Med Chem

StarPEG(PEG200AHNP)2

10 kDaR =

2. Example

AHNP

* p-value calculated by Two-Tailed T-Test = 0.034

In vivo Imaging ofSubcutaneous BT-474 Human Breast Tumor on Nude Mice

P/StarPEG(-PEG200AHNP)2

(2%)/LLL(40%)P/StarPEG(2%)

/LLL(40%)P/PEG200-AHNP(2%)

P/PEG200-AHNP(2%)/LLL (40%)

*p<0.05

Effi

cien

cy (

cm2)

-1E-06

2E-22

1E-06

2E-06

3E-06

4E-06

5E-06

P/StarPEG-(PEG200(AHNP)2

(2%)/LLL(40%)P/StarPEG(2%)

/LLL(40%)P/PEG200

AHNP(2%)P/PEG200

AHNP(2%)/LLL (40%)

Code kon (s-1M-1) koff (10-3 sec-1) Kd(10-6 M)

AHNP 800 0.42 0.52

StarPEG -AHNP2

118 0.54 4.6

StarPEG -AHNP4

1.5 0.62 41

StarPEG-AHNP6

n.d. n.d. n.d.

StarPEG-AHNP8

n.d. n.d. n.d.

Surface Plasmon Resonance kinetics

Surface Plasmon Resonance kinetic parameters Dynamic light scatter (DLS)

Ln Ln-1 + L

L

Rate limiting dissociationof self-aggregate

(1)

L + HER2

HER2-L

(2)

(2)

(1)

StarPEG(AHNP)n

Hydrodynamic diameter (nm)

n = 2

n = 4

n = 8

n = 0

Conclusion:

(1) Polymalic acid is qualified for peptide targeting

(2) Need of appropriate linkers

Syntheses: Hui DingImaging: Pallavi Gangalum

Martz Discovery Fund