Biodegradable nanobrushes for drug delivery

Transcription

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

2 Liposome Nanoparticle Gold Nanoparticle Micelle Third generation of Nano-based arriers For Disease Detection and Therapy Drug Targeting Agent Imaging Agent Linker arbon Nanotube Polymer-onjugate Dendrimer

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

4 Polycefin Functional ore Polymalic acid-trileucine copolymer H-H-H 2 --H H 1 2 H H 5 H 2 H = = n H L-Malic acid H Leu-Leu-LeuH Polymalic acid-trileucine copolymer Disulfide, cleaved by Glutathion in the ytoplasm Targeting Antisense ligonucleotide Prodrug Monoclonal Antibody (mab)

5 ancer cell Late Endosome I.V. Injection Blood vessel Early Endosome Nucleus Glutathione Endocytosis Antisense Mouse TfR Akt P4 EGFR mrna EGFR EGFR signalling Extravasation Active via receptor EPR -effect passive

6 Preparation of nanodrugs for brain tumor treatment H H H Polymalic acid Step-1 H mpeg 5000 Step-2 SH H 3 H 3 H 3 H S S S S S S S S mpeg 5000 S H 3 H 3 HN HN HN PEG 3400 AN-1 AN-2 AN-3 PEG 3400 Alx-680 optional H 3 S S mab-1 mab-2 GBM: hain-α4 hain-β1 ( - ) anti-mstfrmab anti-hutfrmab HER2-positive breast: HER2 Triple-negative breast: EGFR ( - ) ( - ) (anti-mstfrmab) Herceptin ( - ) ( - ) (anti-ms-tfrmab) etuximab

7 Examples for drug delivery by Polycefin variants Primary breast cancer Brain metastases Primary HER2-Positive Breast ancer Metastatic HER2-Positive Breast ancer 47% survival rate improvement 100 Survival % Primary Triple Negative Breast ancer 100 Metastatic Triple Negative Breast ancer 79% survival rate improvement Survival % Days

8 Replacement of antibody by affinity peptide: Pro and ontra Pro Multiple peptides per conjugate Robust structure Smallsize for slender shape of polymeric nanoconjugate Increased diffusibility Deep tissue penetration Reduced immunogenicity Humanization not required Possibility of multivalency hemical fabrication ontra Less affinity to bind to target Absence of Fc and biological activity Less passive tissue targeting (EPR) Low Stability/reduced longevity in plasma Possibility of unscheduled side reactions Tendency for aggregation Easy packaging and delivery. Decreased overall MW of nanodrugandless injectable drug volumen

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

10 Late Endosome I.V. Injection Early Endosome Blood vessel Nucleus Endocytosis LRP-1 Extravasation through BBB into glioblastoma LRP-1 Glioblastoma cell For Uptake and Imaging: P/PEG Angiopep(2%)/AlexaFluor680 (0.5%)

11 Fluorescence Imaging of Glioblastoma-Nude Mouse Model Tumor PMLSA-Angiopep-2: P/PEG2000-Angiopep(2%)/AlexaFluor680

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

13 2. Example AHNP Target: HER2 Ramachandran Murali et al. (2001) J Med hem H H H2 H H H Nanoconjugate kda S S AN (2%) H 3 H 3 H 3 H 3 H 3 H H 3 S N PEG 3400 R S H 3 H 3 N Alexa FluoR 860 (2%) (0.5%) H 3 H 3 H 3 H H 3 LLL(40%) LLL(40%) R = StarPEG(PEG 200 AHNP) 2 10 kda

14 In vivo Imaging of Subcutaneous BT-474 Human Breast Tumor on Nude Mice P/StarPEG(2%) /LLL(40%) P/PEG AHNP(2%) P/PEG AHNP(2%) /LLL (40%) P/StarPEG(-PEG 200 AHNP) 2 (2%)/LLL(40%) 5.E-06 *p< E-06 Efficiency (cm 2 ) 3.E-06 2.E-06 1.E-06 0.E+00 P/StarPEG(2%) /LLL(40%) P/PEG 200 AHNP(2%) P/PEG 200 AHNP(2%) /LLL (40%) P/StarPEG-(PEG 200 (AHNP) 2 (2%)/LLL(40%) * p-value calculated by Two-Tailed T-Test = 0.034

15 Surface Plasmon Resonance kinetics Rate limiting dissociation of self-aggregate (1) (1) L n L L n-1 + L (2) L + HER2 HER2-L (2) StarPEG(AHNP) n Surface Plasmon Resonance kinetic parameters ode k on (s -1 M - 1 ) k off (10-3 sec-1) Kd(10-6 M) AHNP StarPEG- AHNP2 StarPEG- AHNP4 StarPEG- AHNP6 StarPEG- AHNP n.d. n.d. n.d. n.d. n.d. n.d. Dynamic light scatter (DLS) n = 0 n = 2 n = 4 n = 8 Hydrodynamic diameter (nm)

16 onclusion: (1) Polymalic acid is qualified for peptide targeting (2) Need of appropriate linkers Syntheses: Hui Ding Imaging: Pallavi Gangalum Martz Discovery Fund