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

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

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

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

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

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: HER2 ( - ) ( - ) (anti-MsTfRmAb) Herceptin Triple-negative breast: EGFR ( - ) ( - ) (anti-Ms-TfRmAb) Cetuximab

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

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

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

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

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

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

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 250-500 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 =

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

Rate limiting dissociation Ln 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

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