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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
Scheme of targeted polymeric
drug delivery system
Low molecular drug
Targeted polymer drug
Drug
TM





low specificity
side effects (toxicity for healthy tissue)
low bioavailability - rapid excretion
need of repeated dosage
fast degradation - inactivation
Drug
 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
pH-controlled activation
X Enzymatic activation
Binding of Ab or targeting peptides
to polymer carrier
Targeting moieties (TM):
Antibodies (Ab) or their fragments (afinity X characterization, modification, price)
Peptides (synthesis, modification, price X afinity)
 Multivalent covalent
modification of Ab
 Covalent attachment
Drug
TM
Drug
Tumor cell
Possible damage of binding site decrease of binding activity
Normal cell
Specific, non-destructive
Synthesis of peptide sequences
(targeting and CPP)
DIPEA, DCM
1) 20% PIP/DMF
2) Fmoc-aa, PyBOP, HOBt, DIPEA
3) repeat steps 1 and 2
peptide
Fmoc peptide
1) 25% PIP/DMF
2) Fmoc-Peg4 , PyBOP, HOBt, DIPEA
3) 25% PIP/DMF
4) 5-azidopentanoic acid, PyBOP, HOBt
5) TFA
Prepared peptides – TP: GRGDG, c(RGDfK), c(DRfGK)
- CPP: R9F2, PFVYLI, YARAAARQARA
Synthesis of peptide-polymer conjugates
using “click” chemistry catalyzed by Cu(I)
DMSO, AIBN
60 oC, 6 h
HPMA
Dy-633
DMF
peptide
CuSO4, sodium ascorbate
peptide
Dy-633
GRGDG, c(RGDfK), c(DRfGK)
PFVYLI, R9F2
Dy-633
Intensity of fluorescence after
48 hrs of incubation with cells
Mean fluorescence intensity
6000
5000
4000
3000
HUVEC
3T3
SW620
EL-4
2000
1000
0
Control
polymer
2,6
GRGDG
5,1
13,5
c(RGDfK) c(DRfGK)
13,5
12,5
Cells
wt % peptide
CELL CULTURE STUDIES
kinetic flow cytometry (FACS)
Polymer with PFVYLI peptide
Polymer with R9F2 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.
4 °C
Control polymer
Confocal fluorescent microscopy
Control polymer
Polymer with PFVYLI peptide
Polymer with R9F2 peptide
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).
Synthesis of peptide-polymer conjugates
using “click” chemistry with Ru complexes
Pir
X
Pir
DMF
DIPEA
Cu(I)
peptide
CpRuCl(COD)
DMF - Ar
Pir
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
Pir
peptide
Synthesis of peptide-polymer conjugates
using copper-free “click” chemistry
CTA, ABIN
t-BuOH
10% DMSO
24 h, 70°C
HPMA
Ma-GFLG-TT
ABIN
DMSO
2 h, 80°C
Pir
DBCO-NH2
H2O - Ar
methanol
DMA
DMF
DMSO
 Oxygen is not an inhibitor of “click” reaction
 Metal free system, no contamination by catalyst
 Reactive in water, methanol, DMA, DMF and DMSO
Pir
GRGDG, c(RGDfK), c(DRfGK)
peptide PFVYLI, R9F2
Analysis and measuring cell
viability/Cytotoxicity
peptide
wt %
Pir
wt %
IC50
µg/ml
MW
g/mol
IP
HPMA-co-Ma-GFLG-co-MaGFLG-DBCO
0.0
0.0
-
46 000
1.08
HPMA-co-Ma-GFLG-co-MaGFLG-Pirarubicin
0.0
9.0
5.9
41 000
1.15
HPMA-co-Ma-GFLG-DBCO-coMa-GFLG-Pirarubicin
0.0
10.2
3.9
58 000
1.13
HPMA-co-Ma-GFLG-PEG-R9F2
-co-Ma-GFLG-Pirarubicin
10.0
9.3
18.8
58 000
1.17
Pirarubicin
0.0
100
0.06
CONJUGATES
Release of Pirarubicine from polymer incubated in
phosphate buffer pH 7.4 in presence or absence
of cathepsine B
60
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.
Pir released, %
50
40
30
20
10
0
0
10
20
30
40
50
Time, h
without cath B
with cath B
60
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 cells
•with 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
Acknowledgement
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)
Thank you for your attention