1. Polymer drug carriers with enhanced penetration into tumor cells R. Pola, M. Pechar, A. Hoecherl, O. Janoušková, K. Ulbrich Institute of Macromolecular Chemistry, CAS, Prague, Czech Republic

2. Scheme of targeted polymeric drug delivery system Drug Enzymatic activation pH-controlled activation X TM Low molecular drug Targeted polymer drug  low specificity  side effects (toxicity for healthy tissue)  low bioavailability - rapid excretion  need of repeated dosage  fast degradation - inactivation  decrease acute toxicity (no activity during transport)  organ- or cell-specific delivery (targeting)  controlled release (activation at the site of required therapeutic effect)  prolong circulation

3. Binding of Ab or targeting peptides to polymer carrier  Multivalent covalent modification of Ab Possible damage of binding site - decrease of binding activity  Covalent attachment Specific, non-destructive Normal cell Tumor cell Drug TM Targeting moieties (TM): Antibodies (Ab) or their fragments (afinity X characterization, modification, price) Peptides (synthesis, modification, price X afinity)

4. Synthesis of peptide sequences (targeting and CPP) Prepared peptides – TP: GRGDG, c(RGDfK), c(DRfGK) - CPP: R 9 F 2, PFVYLI, YARAAARQARA

5. Synthesis of peptide-polymer conjugates using “click” chemistry catalyzed by Cu(I)

6. 0 1000 2000 3000 4000 5000 6000 Mean fluorescence intensity HUVEC3T3 SW620EL-4 Intensity of fluorescence after 48 hrs of incubation with cells Control GRGDG c(RGDfK) c(DRfGK) Cells polymer 2,6 5,1 13,5 13,5 12,5 wt % peptide

7. CELL CULTURE STUDIES kinetic flow cytometry (FACS) Polymer with PFVYLI peptide Polymer with R 9 F 2 peptide 37 °C The uptake into Jurkat cells over-time (endocytosis inhibited in 4°C). The fluorescence in cells incubated with 0.65 µM control polymer. Control polymer 4 °C

8. Confocal fluorescent microscopy Polymer with PFVYLI peptide Control polymer The rapid visualization of Jurkat cells at the conjugates concentration 1.3 µM. The fluorescence of control polymer was measured as a control; the nuclei were dyed with Hoechst (blue). Polymer with R9F2 peptide

9. Synthesis of peptide-polymer conjugates using “click” chemistry with Ru complexes Ruthenium complex can be used for preparation of conjugates with Pir Ruthenium catalyst is deactivated with air oxygen, Ar atmosphere is necessary Heterogeneous reaction, contamination of the sample by ruthenium Poor solubility of the ruthenium complexes

10. Synthesis of peptide-polymer conjugates using copper-free “click” chemistry Oxygen is not an inhibitor of “click” reaction Metal free system, no contamination by catalyst Reactive in water, methanol, DMA, DMF and DMSO

11. Analysis and measuring cell viability/Cytotoxicity CONJUGATES peptide wt % Pir wt % IC50 µg/ml M W g/mol IP HPMA-co-Ma-GFLG-co-Ma- GFLG-DBCO 0.0 -46 0001.08 HPMA-co-Ma-GFLG-co-Ma- GFLG-Pirarubicin 0.09.05.95.941 0001.15 HPMA-co-Ma-GFLG-DBCO-co- Ma-GFLG-Pirarubicin 0.010.23.93.958 0001.13 HPMA-co-Ma-GFLG-PEG-R9F2 -co-Ma-GFLG-Pirarubicin 10.09.318.858 0001.17 Pirarubicin0.01000.06

12. Release of Pirarubicine from polymer incubated in phosphate buffer pH 7.4 in presence or absence of cathepsine B Pirarubicin atached via GFLG spacer is stable during the circulation in the blood stream and release occurs in the secondary lysosomes of cells due to the attack of the lysosomal enzymes.

13. Summary  Synthesis of targeting and cell-penetrating peptides terminated by azido group using Fmoc solid-phase strategy.  We prepared fluorescently labeled polymers targeted by RGD-based peptides and showed the possibility of targeting these conjugates to HUVEC and 3T3 cellswith CPP and improved the cell penetration to cancer cells. We showed fast uptake to apoptotic cells, no such uptake with control polymer  We demonstrated possibility of using “click” chemistry for conjugation of the unprotected peptide-azides to DBCO-bearing copolymers of p(HPMA) with drug Pir prepared by RAFT polymerization resulting in conjugates with low polydispersity  IC50 value and release of Pir is not influenced by the presence of peptides on polymer backbone

14. Anna Vankova, Michal Pechar, Anita Hoecherl, Olga Janouskova and Karel Ulbrich This work was supported by the Ministry of Education, Youth and Sports of the Czech Republic (grant No. EE2.3.30.0029) Acknowledgement

15. Thank you for your attention