1. Biodegradable nanobrushes for drug delivery
Nanotek & Expo 2014
Biodegradable nanobrushes for drug delivery
Eggehard Holler, Hui Ding, Ramachandran Murali, Julia Y. Ljubimova
Cedars-Sinai Medical Center, Los Angeles, USA

2. Dendrimer Carbon Nanotube Micelle Gold Nanoparticle Nanoparticle
Drug
Targeting Agent
Imaging Agent
Linker
Dendrimer
Carbon Nanotube
Micelle
Gold Nanoparticle
Third generation of
Nano-based Carriers
For Disease Detection
and Therapy
Nanoparticle
Liposome
Polymer- Conjugate

3. Synthesis and degradation
Poly(β-L-malic acid) β α OH
HOOC
n = 500 to 3000
Physarum:
HOOC OH
n = 40 to 80
Aureobasidium:
Depolymerisation:
Spontaneous
& Enzymatic
From
Glucose
Malate
Purification
Plastic bags
Medical
support
Drug delivery
Malignancy
Infection
Contrast agents
Imaging
Nano scale

4. Monoclonal Antibody (mAb)
Polycefin – Functional Core
Polymalic acid-trileucine copolymer 1 2 3 4 5
HO-CH-CH2-C-OH
O=C OH = O O C H C H C O C H C H C O H 2 2 O C O O C O n O H NH
L-Malic acid
Leu-Leu-LeuCOOH
Polymalic acid-trileucine copolymer
Disulfide, cleaved by
Glutathion in the
Cytoplasm
Targeting
Prodrug
Monoclonal Antibody (mAb)
Antisense
Oligonucleotide

5. Cancer cell I.V. Injection  Nucleus Blood vessel Active Extravasation
Anti-EFGR mAb
(Cetuximab)
Anti-mouse TfR mAb
PEG
Antisense RNAEGFR
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
PO4
EPR -effect passive

6. Preparation of nanodrugs for brain tumor treatment
Step-2
Step-1
mAb-1
mAb-2
AON-1
AON-2
AON-3
Polymalic acid
mPEG5000
Alx-680
optional
GBM: Chain-α4 Chain-β1 ( - ) anti-MsTfRmAb anti-HuTfRmAb
HER2-positive breast: HER ( - ) ( - ) (anti-MsTfRmAb) Herceptin
Triple-negative breast: EGFR ( - ) ( - ) (anti-Ms-TfRmAb) Cetuximab

7. 79% survival rate improvement
Examples for drug delivery by Polycefin variants
Primary Triple Negative Breast Cancer
Primary HER2-Positive Breast Cancer
Primary breast cancer
Brain metastases
47% survival rate improvement
Metastatic HER2-Positive Breast Cancer
Survival % 25 50 75
100
79% survival rate improvement
Survival % 10 20 30 40 60 70 80
Days
Metastatic Triple Negative Breast Cancer

8. Replacement of antibody by affinity peptide: Pro and 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 diffusibility
Deep tissue penetration
Low Stability/reduced longevity in plasma
Reduced immunogenicity
Humanization 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

9. Michel Demeule, et al. (2008) J Parm Exp Therapeut
1. Example
Targeting Brain tumor
Angiopep
Michel Demeule, et al. (2008) J Parm Exp Therapeut
Target on BBB endothelial cells: LRP-1
Guangqing X and Liang-Shang G (2013) Int J Cell Biol

10. Angiopep I.V. Injection Nucleus Blood vessel LRP-1 Glioblastoma cell
For Uptake and Imaging: P/PEG2000-Angiopep(2%)/AlexaFluor 680 (0.5%)
Glioblastoma
cell
Late Endosome
Early Endosome
Nucleus
Extravasation through BBB into glioblastoma
I.V. Injection
Blood vessel
Angiopep
PEG2000
P = polymalic acid
AlexaFluore 860
Endocytosis
LRP-1
LRP-1

11. Fluorescence Imaging of Glioblastoma-Nude Mouse Model
Tumor
PMLSA-Angiopep-2: P/PEG2000-Angiopep(2%)/AlexaFluor 680

12. Specific problem with affinity peptides:
Self-association and aggregation because of:
Electrostatic complementation
Lipophilic amino acids

13. 2. Example AHNP R = Nanoconjugate 250-500 kDa StarPEG(PEG200AHNP)2
Target: HER2
Ramachandran Murali et al. (2001) J Med Chem O O O O O O O O O O O C O O C O O C O O C O O C O O C O O C O O C O O C O O C O O H N H N H O H O O H 2 H N H O H N H N H O H O H O C H
Nanoconjugate
kDa 3 S O C H S N H S 3 C H N H 3 C H S 3 O C H O N O O 3 N O O C H 3 A O N N H C H 3 A l e x a N H C H P E G 3
(2%) 3 4 F l u o
R 860 O C H 3 R
(2%)
(0.5%) O C H O H 3 C H O H 3 C H 3
(40%) L L L
(40%) L L L
StarPEG(PEG200AHNP)2
10 kDa
R =

14. Subcutaneous BT-474 Human Breast Tumor on Nude Mice
In vivo Imaging of
Subcutaneous BT-474 Human Breast Tumor on Nude Mice
P/StarPEG(-PEG200AHNP)2
(2%)/LLL(40%)
P/StarPEG(2%)
/LLL(40%)
P/PEG200-AHNP(2%)
/LLL (40%)
*p<0.05
Efficiency (cm2)
P/StarPEG-(PEG200(AHNP)2
(2%)/LLL(40%)
P/StarPEG(2%)
/LLL(40%)
P/PEG200
AHNP(2%)
/LLL (40%)
* p-value calculated by Two-Tailed T-Test = 0.034

15. Rate limiting dissociationLn
Ln-1 + L L
Rate limiting dissociation
of self-aggregate
(1)
L + HER2
HER2-L
(2)
Surface Plasmon Resonance kinetics
(1)
(2)
StarPEG(AHNP)n
Surface Plasmon Resonance kinetic parameters
Dynamic light scatter (DLS)
Hydrodynamic diameter (nm)
n = 2
n = 4
n = 8
n = 0
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.
StarPEG-AHNP8

16. Imaging: Pallavi Gangalum
Conclusion:
Polymalic acid is qualified for
peptide targeting
(2) Need of appropriate linkers
Syntheses: Hui Ding
Imaging: Pallavi Gangalum
Martz Discovery Fund