Nanotek & Expo 2014 - PowerPoint Presentation

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Nanotek

& Expo 2014Biodegradable nanobrushes for drug delivery

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

Cedars-Sinai Medical

Cen

ter

,

Los

Angeles

, U

SA

Drug

Targeting AgentImaging Agent

Linker

Dendrimer

Carbon Nanotube

Micelle

Gold Nanoparticle

Third generation

of

Nano-based

C

arriers

For Disease Detection

and T

herapy

Nanoparticle

Liposome

Polymer- Conjugate

Synthesis and degradation

From

Glucose

OH

HOOC

n = 500 to 3000

Physarum

:

HOOC

OH

n = 40 to 80

Aureobasidium

:

Poly(

β

-

L

-malic acid)

Depolymerisation

:

Spontaneous

& Enzymatic

Malate

Purification

Plastic bags

Medical

support

Drug delivery

Malignancy

Infection

Contrast agents

Imaging

Nano scale

α

β

Antisense

Oligonucleotide

Monoclonal

Antibody (

mAb

)

Disulfide, cleaved by

Glutathion in the

Cytoplasm

Polymalic

acid-

trileucine

copolymer

Prodrug

Targeting

O

C

H

1

C

H

2

2

C

O

O

H

C

3

O

O

4

C

H

5

C

H

2

C

O

NH

C

O

H

O

n

Leu-Leu-

Leu

CO

OH

Polymalic

acid-

trileucine

copolymer

Polycefin – Functional Core

HO-CH-CH

2

-C-OH

O=C

OH

=

O

L

-Malic acid

Anti-EFGR

mAb

(

Cetuximab

)

Anti-mouse

TfR

mAb

PEG

Antisense RNA

EGFR

Cancer cell

Late

Endosome

Early

Endosome

Gluta-thione



Nucleus

Akt

Antisense

EGFR

mRNA

EGFR

signalling

Endocytosis

EGFR

Active

via receptor

Extravasation

Mouse

TfR

I.V. Injection

Blood vessel

PO

4

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

Step-2

Step-1

mAb-1

mAb-2

AON-1

AON-2

AON-3

Polymalic acid

mPEG

5000

mPEG

5000

Alx-680optional

Primary Triple N

egative Breast Cancer

Primary

HER2-Positive

B

reast Cancer

Primary breast cancer

Brain metastases

47

% survival rate improvement

Metastatic

HER2-Positive

B

reast

C

ancer

Survival

%

0

25

50

75

100

0

50

100

79%

survival rate

improvement

S

urvival %

0

10

20

30

40

50

60

70

80

0

50

100

Days

Metastatic

Triple

N

egative

B

reast

C

ancer

Examples for drug delivery by Polycefin variants

Replacement of antibody by affinity peptide: Pro and Contra

ProContraMultiple peptides per conjugateLess affinity to bind to targetRobust structureAbsence of Fc and biological activitySmall size for slender shape of polymeric nanoconjugate

Less passive tissue targeting (EPR)Increased diffusibilityDeep tissue penetrationLow Stability/reduced longevity in plasma

Reduced immunogenicity

Humanization

not required

Possibility of unscheduled side reactions

Possibility of

multivalency

Tendency for

a

ggregation

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/PEG

2000

-Angiopep(2%)/

AlexaFluor

680 (0.5%)

Glioblastoma

cell

Late

Endosome

Early

Endosome

Nucleus

Extravasation through

BBB into glioblastoma

I.V. Injection

Blood vessel

Angio

p

ep

PEG

2000

P =

polymalic

acid

AlexaFluore

860

Endocytosis

LRP-1

LRP-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 complementationLipophilic amino acids

O

H

O

H

N

H

O

C

H

3

C

H

3

N

H

S

N

H

N

H

S

S

A

O

N

R

L

L

L

L

L

L

O

H

N

H

O

C

H

3

C

H

3

N

H

O

C

H

3

C

H

3

O

H

N

H

O

C

H

3

C

H

3

N

C

O

O

O

C

O

O

O

C

O

O

O

C

O

O

O

C

O

O

O

C

O

O

O

C

O

O

O

C

O

O

O

C

O

O

O

C

O

O

O

P

E

G

3

4

0

0

N

O

O

S

N

O

O

A

l

e

x

a

F

l

u

o

R 860

H

O

C

H

3

C

H

3

N

H

O

C

H

3

C

H

3

O

H

O

H

O

H

O

H

O

H

2

Nanoconjugate

250-500

kDa

(40%)

(40%)

(2%)

(0.5%)

(2%)

Target: HER2

Ramachandran Murali et al. (2001) J Med

Chem

StarPEG

(PEG

200

AHNP

)

2

10

kDa

R =

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(-PEG

200

AHNP)

2

(

2

%)/

LLL(40%)

P/

StarPEG

(2%)

/

LLL(40%)

P/PEG

200

-AHNP(2

%)

P/PEG

200

-AHNP(2%)

/

LLL (40%)

*p<0.05

Efficiency (cm

2)

P/

StarPEG

-(PEG

200

(AHNP)2

(

2

%)/

LLL(40%)

P/

StarPEG

(2%)

/

LLL(40%)

P/PEG

200

AHNP(2

%)

P/PEG

200

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

-AHNP

2

118

0.54

4.6

StarPEG

-AHNP4

1.5

0.62

41

StarPEG-AHNP6

n.d.n.d.n.d.StarPEG-AHNP8n.d.

n.d.

n.d.

Surface Plasmon Resonance kinetics

Surface Plasmon Resonance kinetic parameters

Dynamic light scatter (DLS)

L

n

L

n-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: Polymalic acid is qualified for peptide targeting(2) Need of appropriate linkers

Syntheses: Hui DingImaging: Pallavi GangalumMartz Discovery Fund