Biodegradable star HPMA polymer conjugates of doxorubicin for passive tumor targeting

Article abstract of DOI:10.1016/j.ejps.2011.03.001

New biodegradable star polymer-doxorubicin (Dox) conjugates designed for passive tumor targeting were investigated and the present study described their synthesis, physico-chemical characterization, drug release and biodegradation. In the conjugates the core formed by poly(amido amine) (PAMAM) dendrimers was grafted with semitelechelic N-(2-hydroxypropyl)methacrylamide (HPMA) copolymers bearing doxorubicin attached by hydrazone bonds, which enabled intracellular pH-controlled drug release, or by a GFLG sequence, which was susceptible to enzymatic degradation. The controlled synthesis utilizing semitelechelic copolymer precursors facilitated preparation of biodegradable polymer conjugates in a broad range of molecular weights (110-295 kDa) while still maintaining low polydispersity (～1.7). The polymer grafts were attached to the dendrimers either through stable amide bonds or enzymatically or reductively degradable spacers, which enabled intracellular degradation of the high molecular weight polymer carrier to products that were able to be excreted from the body by glomerular filtration. Biodegradability tests showed that the rate of degradation was much faster for reductively degradable conjugates (completed within 4 h) than the degradation of conjugates linked via an enzymatically degradable oligopeptide GFLG sequence (within 72 h). This finding was likely due to the difference in steric hindrance for the small molecule glutathione and the enzyme cathepsin B. As for drug release, the conjugates were fairly stable in buffer at pH 7.4 (model of blood stream) but released doxorubicin either under mild acidic conditions or in the presence of lysosomal enzyme cathepsin B, both of which modeled the tumor cell microenvironment.

Full text of DOI:10.1016/j.ejps.2011.03.001

European Journal of Pharmaceutical Sciences 42 (2011) 527–539
Contents lists available at ScienceDirect
European Journal of Pharmaceutical Sciences
journal homepage: www.elsevier.com/locate/ejps
Biodegradable star HPMA polymer conjugates of doxorubicin for
passive tumor targeting
∗
ˇ
Tomásˇ Etrych , Jirˇí Strohalm, Petr Chytil, Peter Cernoch, Larisa Starovoytova,
Michal Pechar, Karel Ulbrich
Institute of Macromolecular Chemistry, Academy of Sciences of the Czech Republic, v.v.i., Heyrovsky´ Sq. 2, 162 06 Prague 6, Czech Republic
a r t i c l e i n f o
a b s t r a c t
Article history:
New biodegradable star polymer–doxorubicin (Dox) conjugates designed for passive tumor targeting
were investigated and the present study described their synthesis, physico-chemical characterization,
drug release and biodegradation. In the conjugates the core formed by poly(amido amine) (PAMAM)
dendrimers was grafted with semitelechelic N-(2-hydroxypropyl)methacrylamide (HPMA) copolymers
bearing doxorubicin attached by hydrazone bonds, which enabled intracellular pH-controlled drug
release, or by a GFLG sequence, which was susceptible to enzymatic degradation. The controlled syn-
thesis utilizing semitelechelic copolymer precursors facilitated preparation of biodegradable polymer
conjugates in a broad range of molecular weights (110–295 kDa) while still maintaining low polydis-
persity (∼1.7). The polymer grafts were attached to the dendrimers either through stable amide bonds
or enzymatically or reductively degradable spacers, which enabled intracellular degradation of the high
molecular weight polymer carrier to products that were able to be excreted from the body by glomeru-
lar filtration. Biodegradability tests showed that the rate of degradation was much faster for reductively
degradable conjugates (completed within 4 h) than the degradation of conjugates linked via an enzymat-
ically degradable oligopeptide GFLG sequence (within 72 h). This finding was likely due to the difference
in steric hindrance for the small molecule glutathione and the enzyme cathepsin B. As for drug release,
the conjugates were fairly stable in buffer at pH 7.4 (model of blood stream) but released doxorubicin
either under mild acidic conditions or in the presence of lysosomal enzyme cathepsin B, both of which
modeled the tumor cell microenvironment.
Received 19 November 2010
Received in revised form 28 January 2011
Accepted 1 March 2011
Available online 8 March 2011
Keywords:
Dendrimer
HPMA copolymers
Doxorubicin
Drug conjugates
Tumor targeting
© 2011 Elsevier B.V. All rights reserved.
1. Introduction
selection of the polymer carrier, the spacer between the drug and
carrier and, if applicable, the targeting moiety and its binding pro-
Recently, many water-soluble synthetic polymers have been
intensively studied as drug carrier systems. Some of these poly-
mers have become useful drug carriers (Kratz et al., 2008; Pasut and
cedure are all crucial for obtaining a tumor- or tumor cell-specific
drug release, which is important for subsequent antitumor activity
of the drug (Haag and Kratz, 2006; Kopecˇek et al., 2001; Ulbrich
ˇ
ˇ
Veronese, 2009; Ulbrich and Subr, 2010) for the delivery of a num-
and Subr, 2004). The drug can be released from the conjugate by
ber of therapeutics, including anticancer and anti-inflammatory
agents. Conjugation of drugs with water-soluble polymers usually
reduces their toxicity, improves their solubility, bioavailability and
stability (enzymatic, thermal), eliminates undesirable interactions
a very specific enzymatic cleavage (Putnam and Kopecˇek, 1995;
Ulbrich et al., 1996) (e.g., degradation of the oligopeptide sequence
by lysosomal enzymes) or by pH-controlled chemical hydrolysis
ˇ
(Kratz et al., 1999; Rodrigues et al., 2006; Ulbrich and Subr, 2004)
ˇ
in the body, and delays blood clearance (Kratz et al., 2008; Ríhová
(hydrazone bond, cis-aconityl spacer). Among synthetic polymer
drug carriers, the water-soluble HPMA copolymers (Duncan, 2003;
Kopecˇek et al., 2000; Ulbrich et al., 2000) have been the most inten-
sively studied and most promising systems for enabling actively or
passively targeted drug delivery (Etrych et al., 2008a; Ulbrich et al.,
2003; Wang et al., 2000).
et al., 2000; Ulbrich et al., 2000). The coupling of hydrophobic drugs
to water-soluble polymers allows the preparation of hydrophilic
polymer–drug conjugates that retain the activity of the drug.
Polymer–drug conjugates also facilitate specific delivery to the tar-
get tissue and controlled drug release within the tissue. The proper
The treatment potential of the polymeric carrier systems can
be significantly improved by connecting them with targeting moi-
eties, such as antibodies (Etrych et al., 2009; Kovárˇ et al., 2003)
or oligopeptides (Pike and Ghandehari, 2010; Pola et al., 2009), or
∗
Corresponding author. Tel.: +420 296 809224; fax: +420 296 809410.
E-mail address: etrych@imc.cas.cz (T. Etrych).
0928-0987/$ – see front matter © 2011 Elsevier B.V. All rights reserved.
doi:10.1016/j.ejps.2011.03.001

528
T. Etrych et al. / European Journal of Pharmaceutical Sciences 42 (2011) 527–539
by increasing their molecular weight (Dvorˇák et al., 1999; Etrych
et al., 2008a; Wang et al., 2000). Polymer carriers with a molecu-
lar weight above the renal threshold prevent the drug from rapid
blood clearance and elimination from the body. Moreover, the high
molecular weight of the polymer carrier enhances accumulation of
the prodrug in solid tumor tissue due to a phenomenon charac-
terized as the enhanced permeability and retention (EPR) effect
(Maeda, 2001). The EPR effect significantly increases with increas-
ing molecular weight of the delivery system. The effect of EPR on
accumulation of poly(HPMA) in sarcoma (Seymour et al., 1995) and
HPMA copolymers in lymphoma (Etrych et al., 2008a,b) and other
tumors (Steyger et al., 1996) has been demonstrated in mice.
Most synthetic polymers used for drug delivery purposes,
including HPMA copolymers, have a non-degradable main chain.
Polymers with a molecular weight under the renal threshold (e.g.,
approximately 50,000 g/mol for HPMA copolymers) could be elim-
inated by glomerular filtration. Polymer carriers with a molecular
weight above the threshold cannot be easily removed in this way,
and they tend to accumulate in the body. To avoid this unde-
sired accumulation, the high molecular weight (HMW) polymer
drug carriers should be biodegradable or should involve biodegrad-
able spacers, which would enable degradation of the polymer
into fragments that could be eliminated from the organism. Some
biodegradable HMW drug conjugates based on HPMA (Etrych
et al., 2001, 2008a; Shiah et al., 2001) or PEG (Pechar et al., 2005)
were prepared and tested for anticancer activity against selected
tumors.
Here, we describe the synthesis, physico-chemical properties
and preliminary data of bio-chemical evaluation of the new star
HPMA copolymer Dox conjugates. These conjugates were designed
for efficient passive tumor targeting and subsequent biodegrada-
tion, which enabled polymer carrier elimination from the body. The
drug (Dox) was attached to the polymer graft by a hydrazone bond,
which was susceptible to pH-activated hydrolysis enabling Dox
release in a mildly acidic environment (pH 5–5.5), or an oligopep-
tide GFLG sequence, which was tailored for intracellular hydrolysis
by lysosomal enzymes. The star polymer carriers were prepared by
grafting semitelechelic HPMA copolymers (Mw ∼ 25–45,000 g/mol)
onto polyamidoamine (PAMAM) dendrimer cores, which formed
HMW star-like structures with a narrow distribution of molecu-
lar weights. Proposed star polymer carriers could be synthesized
as non-degradable or bio-degradable long-circulating carriers. The
latter should be degraded after fulfillment of their targeting mis-
sion to solid tumors. The degradability was mediated by spacers
that were susceptible to either enzymolysis or reduction, which
would both enable disintegration of the HMW star conjugate to
the short polymer fragments that could be cleared from the body
by glomerular filtration.
2. Experimental
2.1. Chemicals
Several studies have described that synthetic water-soluble
HPMA copolymers containing anti-cancer drugs (e.g., Dox, cis-
platin, dexamethasone, docetaxel) bound via a hydrolytically
degradable hydrazone bond or an oligopeptide lysosomotropic
sequence resulted in 100% long-term survival of tumor-inoculated
mice (Etrych et al., 2010; Gianasi et al., 1999; Kopecˇek and
Kopecˇková, 2010; Krakovicˇová et al., 2009; Omelyanenko et al.,
1-Aminopropan-2-ol,
methacryloyl
(AIBN),
chloride,
2,2ꢀ-azobis(isobutyronitrile)
4,4ꢀ-azobis(4-
cyanovaleric acid) (ABIC), 6-aminohexanoic acid (ah),
N,Nꢀ-dimethylformamide (DMF), glutathione, benzotriazol-
1-yl-oxytripyrrolidinophosphonium
hexafluorophosphate
(DCC),
N,Nꢀ-
1-hydroxy benzotriazole
(PyBOP),
N,Nꢀ-dicyclohexylcarbodiimide
diisopropylcarbodiimide
(DIPC),
ˇ
1996; Ríhová et al., 2001; Ulbrich et al., 2004). Most of these studies
(HOBT), leucylglycine, glycylphenylalanine, phthalaldehyde
(OPA), N-ethyldiisopropylamine (EDPA), dimethyl sulfox-
ide (DMSO), Fmoc-Gly-OH, Fmoc-Leu-OH, Fmoc-Phe-OH,
were based on linear HPMA copolymers with a molecular weight
under the renal threshold. Later on, we have described branched
and grafted water-soluble polymers with molecular weights above
the renal threshold (Etrych et al., 2008a; Ulbrich et al., 2003).
Increasing the molecular weights of the conjugates improved the
therapeutic effects due to a more pronounced EPR effect. The
drawbacks of the branched and grafted polymer systems are high
polydispersity and lower reproducibility of their synthesis, which
is difficult to overcome during the synthesis of the polymer carriers.
Recently, monodisperse dendrimer molecules, with a shell formed
by various functional groups, were synthesized and investigated for
their use in drug and gene delivery (Dutta et al., 2010; Nishiyama
et al., 2009). Dendrimers offer some advantages over other polymer
conjugates by creating a well-defined, nearly monodisperse drug
carrier scaffold. The use of dendrimers as drug carriers has been
limited by toxicity, which is influenced by the type of repeated
building blocks that form the dendrimer, the dendrimer gener-
ation and the character of the surface groups (McNerny et al.,
2010). The conjugation of synthetic polymers (e.g., HPMA copoly-
mers or PEG) with dendrimers can decrease dendrimer toxicity and
allow the preparation of HMW polymer carriers with low poly-
dispersity (Hedden and Bauer, 2003; Wang et al., 2000). These
polymer-modified dendrimers, however, were non-biodegradable,
which limited their use in human therapy because of undesired
accumulation of the carrier in the body. Moreover, only short poly-
mer grafts (Mw ∼ 5–6000 g/mol) were used for preparation of HMW
constructs thus dendrimers of higher generations had to be applied.
This resulted, due to the steric hindrance reason, in an increase in
remaining free and possibly toxic functional groups on the den-
drimer surface.
Fmoc-Gly-OH,
tert-butyl
carbazate,
4,5-dihydrothiazole-
2-thiol, ethylenediaminetetraacetic acid (EDTA), reduced
glutathione (GSH), ethylenediamine (EDA), N-succinimidyl
3-(2-pyridyldithio)propionate (SPDP), cathepsin
oroacetic acid (TFA) were purchased from Fluka. Doxorubicin
hydrochloride (Dox.HCl) was purchased from Meiji Seiko, Japan
and 2,4,6-Trinitrobenzene-1-sulfonic acid (TNBSA) from Serva,
Heidelberg, Germany. 2-(2-Pyridyldithio)ethylamin hydrochlo-
rid (PDEA) was synthesized according to (Shiah et al., 2001).
Poly(amido amine) (PAMAM) dendrimers were purchased from
Dendritic Nanotechnologies, Inc., USA.
B and triflu-
2.2. Synthesis of monomers, oligopeptide and modified dendrimer
N-(2-Hydroxypropyl)methacrylamide (HPMA) was synthesized
as described in Ulbrich et al. (2000) using K2CO3 as a base. M.p.
70 ◦C; purity >99.8% (HPLC); elemental analysis: calcd., C 58.72%, H
9.15%, N 9.78%; found, C 58.98%, H 9.18%, N 9.82%.
N-(tert-Butoxycarbonyl)-Nꢀ-(6-methacrylamidohexanoyl)
hydrazine (Ma-ah-NHNH-Boc) was prepared in two-step synthe-
sis as described in Ulbrich et al. (2004). M.p. 110–114 ◦C; purity
(HPLC) >99.5%; elemental analysis: calcd., C 57.70 C, H 8.33, N
13.46; found, C 57.96, H 8.64, N 13.25.
6-Methacrylamidohexanohydrazide (Ma-ah-NHNH2) was pre-
pared in two-step synthesis as described in Etrych et al. (2008b).
M.p. 79–81 ◦C; elemental analysis: calc. C 56.32%, H 8.98%, N
19.70%; found C 56.49%, H 8.63%, N 19.83%.

T. Etrych et al. / European Journal of Pharmaceutical Sciences 42 (2011) 527–539
529
N-Methacryloylglycyl-dl-phenylalanyl-l-leucylglycine
4-
25% piperidine in DMF. Cleavage of the unprotected peptide H-
Gly-Phe-Leu-Gly-OH from the resin was performed with solution
95% trifluoracetic acid (TFA), 2.5% triisopropylsilane and 2.5%
H2O (25 mL/g of resin), for 2 h. The resin was filtered off and
rinsed with TFA. The filtrate was concentrated under vacuum and
precipitated with diethyl ether. The precipitate was isolated by
filtration and dried in vacuum yielding 0.52 g (88%) of the pep-
tide derivative. The product was characterized by MALDI-TOF MS
(392.5 M+H) and reverse-phase high-performance liquid chro-
matography (HPLC) showing single peak with a retention time
of 18.0 min.
PAMAM dendrimer with pyridyldisulfanyl groups (D-PDS;
Fig. 2) was prepared by the reaction of amino groups of PAMAM
dendrimer (G2, cystamine core, 16 amino groups) with SPDP in
methanol as follows: to a solution of 5 mg PAMAM dendrimer
(0.023 mmol amino groups) in 0.5 mL methanol was under gen-
tle stirring added solution of 23 mg SPDP (0.072 mmol) in 0.5 mL
methanol. After 2 h of stirring low-molecular-weight (LMW) impu-
rities were removed by gel filtration on Sephadex LH-20 column
(50 cm × 3 cm, 260 nm UV detection) using methanol as eluent.
Product was stored in methanol solution (10 wt%) at −18 ◦C. Yield:
5.8 g (66%). The product was characterized by number of PDS
groups and HPLC showing single peak with a retention time of
10.79 min. Chemical structure of the dendrimers was determined
by NMR spectrometer. 1H and 13C NMR spectra of 5% (w/v) DMSO-
d6 or MeOD solution were measured with a Bruker 600MH Avance
nitrophenyl ester (Ma-GFLG-ONp) was synthesized as previously
reported (Ulbrich et al., 2000). M.p. 134–136 ◦C; amino acid
analysis: Gly/L-Phe/D-Phe/L-Leu = 2.05/0.54/0.47/1.00; elemental
analysis calcd., C 59.89%, H 6.07%, N 12.04%; found, C 59.21%, H
6.25%, N 12.32%; HPLC showed two peaks of almost equal areas at
14.41 min (l-Phe peptide) and 14.71 min (d-Phe peptide).
(N-Methacryloylglycyl)-dl-phenylalanylleucylglycyldoxorubicin
(Ma-GFLG-Dox) was prepared by the reaction of an equiva-
lent amount of Ma-GFLG-ONp and Dox.HCl in DMF at 4 ◦C as
previously reported (Ulbrich et al., 2000). TLC: two spots at
Rf = 0.46 and 0.4 corresponding to the d-Phe and L-Phe iso-
mers (chloroform–methanol 8:1), HPLC showed a single peak at
18.22 min (UV detection at 230 and 484 nm), amino acid analysis:
Gly/L-Phe/D-Phe/L-Leu 2.06:0.56:0.45:1.00.
The linear tetrapeptide H-Gly-Phe-Leu-Gly-OH (H-GFLG-OH)
was assembled by the automatic solid phase peptide synthesis
using automatic peptide synthesizer, starting from the C-terminus
using standard Fmoc procedures, by consecutive addition of the N-
Fmoc-protected amino acid (2.5 equiv.), PyBOP (2.5 equiv.), HOBt
(2.5 equiv.), and EDPA (5.0 equiv.) in DMF. Amino acid derivatives
were coupled in the following order: Fmoc-Gly-OH, Fmoc-Leu-OH,
Fmoc-Phe-OH, Fmoc-Gly-OH. Fmoc groups were removed using
piperidine-DMF (1:4). The loading of the 2-chlorotrityl chloride
resin with Fmoc-Gly-OH was determined by spectrophotomet-
ric measurement of the Fmoc group (1.5 mmol/g) released with
+
O
O
R
HN
HO
ABIC-TT
DMSO,
60°C, 6h
CN
CN
GFLG OH
H
GFLG OH
NH
N
S
y
O
x
y
x
O
O
O
O
O
S
R
R
HN
HO
HN
HO
P(Hy)-TT and P(DoxA)-TT
P(Hy)-OH
PDEA
CN
for P(Hy)-TT;-PDS;-SH;-OH
NH
S
N
O
S
y
O
x
O
NH
HN
NH
O
R =
O
5
R
HN
HO
O
for P(DoxA)-TT;-PDS;-SH
P(Hy)-PDS and P(DoxA)-PDS
DTT
CN
GFLG NH
OH
HN
CH3
O
O
OH
NH
SH
R =
y
O
x
O
O
O
O
R
HN
HO
HO
O
HO
P(Hy)-SH and P(DoxA)-SH
OH
O
Fig. 1. Schematic description of synthesis of semitelechelic polymer precursors.

530
T. Etrych et al. / European Journal of Pharmaceutical Sciences 42 (2011) 527–539
R1HN
NHR1
NHR1
R1HN
NH
O
HN
O
HN
NH
O
O
O
O
N
N
NHR1
R1HN
O
O
O
NH
NH
HN
HN
NH
HN
N
N
HN
O
NH
O
N
N
O
O
O
O
O
NHR1
NHR1
NH
NH
R1HN
R1HN
HN
N R N
2
HN
O
O
O
O
O
N
O
N
NH
NH
HN
HN
HN
NH
N
N
O
NH
HN
O
NHR1
R1HN
N
N
O
O
O
O
HN
NH
NH
O
HN
NHR1
R1HN
R1HN
NHR1
; R2 = (CH2)2SS(CH2)2
D-NH2 R1 = H; R2 = (CH2)4
S
N
D-PDS R1 =
S
Fig. 2. Structure of dendrimers used in the synthesis of polymer conjugates: amino groups-containing dendrimer D-NH2 and pyridyldisulfanyl groups containing dendrimer
D-PDS.
III spectrometer (5 mm NMR tubes were used). The integrated
intensities were determined with the spectrometer integration
software with accuracy of 1%. The temperature was maintained
constant within 0.2 K with BVT 3000 temperature unit. HMDSO
was used as internal standard. Typical measurement condition for
1H were as follows: spectral width 6 kHz, pulse width 10 ␮s (90◦
pulse), relaxation delay 20 s, 64 scans at temperature 300 K. Typical
measurement conditions for 13C NMR measurements were as fol-
lows: 150 MHz operating frequency, relaxation delay 20 s, 90◦ pulse
width 8 ␮s, spectral width 36 kHz, 6000 scans. COSY experiment:
spectral width 6 kHz, pulse width 10 ␮s (90◦ pulse), relaxation
delay 20 s, 64 scans, TD 1024, at temperature 300 K.
Purity of all the monomers, oligopeptide and D-PDS mentioned
above was examined by HPLC (Shimadzu, Japan) using a reverse-
phase column Chromolith Performance RP-18e 100-4.6 with UV
detection at 230 nm, eluent water–acetonitril with acetonitril gra-
dient 0–100 vol.%, flow rate 0.5 mL/min.
The content of end-chain TT groups was determined spectropho-
tometrically on a Helios ␣ (Thermochrom) spectrophotometer
using ε305 = 10,700 L mol−1 cm−1 (methanol) (Subr and Ulbrich,
ˇ
2006).
The TT-terminated semitelechelic HPMA copolymer containing
Dox attached via amide bond to oligopeptide GFLG spacer (poly-
mer P(DoxA)-TT, Table 1) was prepared by radical copolymerization
in DMSO initiated with ABIC-TT. HPMA (300 mg, 2.1 mmol), Ma-
GFLG-Dox (90 mg, 0.09 mmol) and ABIC-TT (23 mg, 0.047 mmol)
were dissolved in DMSO (2.2 mL). The solution was introduced into
an ampoule, bubbled with nitrogen, and sealed. The polymeriza-
tion was carried out at 60 ◦C for 6 h. The polymer was isolated
by precipitation into an acetone–diethyl ether mixture (2:1) and
purified by reprecipitation from methanolic solution into the same
mixture. The polymer was filtered off, washed with diethyl ether,
and dried in vacuum. The yield was 0.24 g (62.8%). The content of
thiazolidine-2-thione (TT) groups was determined spectrophoto-
metrically as mentioned above.
The 2-pyridyldisulfanyl (PDS)-terminated semitelechelic HPMA
copolymers containing either Boc-protected hydrazide groups
(polymer P(Hy)-PDS, Table 1) or containing Dox attached via amide
bond to oligopeptide GFLG spacer (polymer P(DoxA)-PDS, Table 1)
were prepared by the reaction of terminal TT groups of the polymer
P(Hy)-TT, resp. P(DoxA)-TT, with 2-(2-pyridyldithio)ethylamine
hydrochloride in DMF. Example of the reaction: PDEA (16 mg,
0.072 mmol) and N-ethyldiisopropylamine (12 ␮L) were dissolved
in DMF (0.6 mL) and a solution of polymer P(DoxA)-TT (600 mg,
0.056 mmol TT) in 3.5 mL DMF was added. After 3 h of stirring the
reaction mixture was diluted with methanol to reach 10 mL in total
and LMW impurities were removed by gel filtration on Sephadex
LH-20 column using methanol as eluent. The copolymer was iso-
lated by precipitation into ethyl acetate. Content of PDS groups was
determined by UV spectrophotometry after reaction with dithio-
threitol (Ulbrich et al., 2003).
2.3. Synthesis of polymer precursors and polymer–drug
conjugates
The thiazolidine-2-thione (TT)-terminated semitelechelic
HPMA copolymer containing tert-butoxycarbonyl (Boc)-protected
hydrazide groups (polymer P(Hy)-TT, Table 1) was prepared by
radical solution copolymerization of HPMA (2.0 g, 0.014 mol)
with Ma-ah-NHNH-Boc (378 mg, 1.21 mmol) in DMSO (14.4 mL)
initiated with azo-initiator 3,3ꢀ-[azobis(4-cyano-4-methyl-1-
oxobutane-4,1-diyl)]bis(thiazolidine-2-thione) (ABIC-TT, 0.76 g,
ˇ
1.57 mmol) as described (Subr and Ulbrich, 2006). Polymerization
was carried out in a sealed ampoule at 60 ◦C (6 h). The polymer
was isolated by precipitation into acetone–diethyl ether mixture
(1:1) and purified by re-precipitation from methanol solution
into the same mixture. The polymer was filtered off, washed with
diethyl ether, and dried in vacuum. The yield was 1.71 g (72.8%).

T. Etrych et al. / European Journal of Pharmaceutical Sciences 42 (2011) 527–539
531
Table 1
Characteristics of semitelechelic polymer precursors.
a
b
Polymer
Mw (g/mol)
Mw/Mn
Hydrazide (Hy) content (mol%)
Dox contentd (wt%)
–
Chain end-group
Mn,T (g/mol)
11,000
Mn/Mn,T
P(Hy)-TT
26,500
41,200
29,500
42,600
31,700
43,500
29,500
27,500
1.84
1.74
1.72
1.80
1.83
1.85
1.87
1.79
5.4
–
5.4
–
5.5
–
5.3
6.6
TT
TT
PDS
PDS
SH
SH
COOH
–
1.31
1.28
1.10
1.14
1.05
1.12
1.05
–
P(DoxA)-TT
P(Hy)-PDS
P(DoxA)-PDS
P(Hy)-SH
10.9
18,500
15,300
20,200
16,800
20,500
15,200
–
–
10.8
–
10.8
–
P(DoxA)-SH
P(Hy)-COOH
P(Hy)c
–
a
Mn,T is number-average molecular weight calculated from the GFLG-OH, TT, SH or PDS group content.
Mn/Mn,T is functionality of a polymer.
Statistical copolymers of HPMA with Ma-ah-NHNH2.
b
c
d
Dox attached via amide bond to oligopeptide GFLG spacer.
The sulfhydril (–SH) group-terminated semitelechelic polymer
or G3, diaminobutan core) in methanol. For example, polymer
P(Hy)-TT (327 mg; 0.03 mmol TT groups) was dissolved in 8 mL of
methanol and added into a stirring solution of 8 mg of D-NH2 (G2,
diaminobutan core, 16 amino groups; 0.002 mmol of dendrimer) in
3.1 mL of methanol. After 2 h, the reaction was finished by adding
5 ␮L of 1-aminopropan-2-ol. Low molecular weight (LMW) impu-
rities were removed by gel filtration (Sephadex LH-20, solvent
methanol). The polymer-modified dendrimer was isolated by pre-
cipitation in ethyl acetate.
precursors, HPMA copolymers containing either Boc-protected
hydrazide groups (polymer P(Hy)-SH, Table 1) or containing Dox
attached via amide bond to oligopeptide GFLG spacer (polymer
P(DoxA)-SH, Table 1) were prepared by reduction of chain-
terminating PDS groups of polymer P(Hy)-PDS or P(DoxA)-PDS
with DTT. Example of the reaction: Polymer P(Hy)-PDS (100 mg,
6.5 ␮mol PDS groups) was dissolved in 2 mL of distilled water and
DTT (15 mg, 97 ␮mol) was added under gentle stirring. After 30 min
LMW impurities were removed by gel filtration using a PD-10
column with distilled water elution. The corresponding polymer
fraction was collected and separated by freeze-drying.
The semitelechelic polymer precursor (polymer P(Hy)-COOH,
Table 1), HPMA copolymer with chain-terminating carboxyl group
of GFLG oligopeptide was prepared by the reaction of terminal
TT groups of polymer P(Hy)-TT with amino group of H-GFLG-
OH oligopeptide in DMF. H-GFLG-OH (85 mg, 0.21 mmol) and
N-ethyldiisopropylamine (100 ␮L) were dissolved in DMF (6 mL)
and a solution of polymer P(Hy)-TT (510 mg, 0.046 mmol TT) in
6 mL DMF was added. After 20 h of stirring, the reaction was fin-
ished by adding 5 ␮L of 1-aminopropan-2-ol, the reaction mixture
was concentrated to 4 mL and diluted with methanol to reach
14 mL in total and LMW impurities were removed by gel filtra-
tion on Sephadex LH-20 column using methanol as eluent. The
copolymer was isolated by precipitation into acetone. Content of
end chain GFLG sequences was determined by amino acid analysis
(Shimadzu, Japan, pre-column OPA derivatization).
Polymer conjugate D2-P(DoxA) (Table 2) containing Dox
attached via amide bond was prepared by aminolysis of TT
groups of semitelechelic HPMA copolymer P(DoxA)-TT with amino
groups of PAMAM dendrimers (D-NH2;G2, diaminobutan core) in
methanol. Polymer P(DoxA)-TT (100 mg; 0.01 mmol TT groups) was
dissolved in 3 mL of methanol and added into a stirring solution of
2.8 mg of D-NH2 (0.7 ␮mol of dendrimer) in 1 mL of methanol. After
3 h, the reaction was finished by adding 3 ␮L of 1-aminopropan-
2-ol. LMW impurities were removed by gel filtration (Sephadex
LH-20) in methanol. The polymer-modified dendrimer was isolated
by precipitation in acetone.
Polymer precursor D2-SS-P(Hy) and conjugate D2-SS-P(DoxA)
(Table 2) were prepared by the reaction of -SH groups of semit-
elechelic polymer P(Hy)-SH (used for polymer D2-SS-P(Hy)) or
P(DoxA)-SH (used for conjugate D2-SS-P(DoxA)) with PDS groups
of the D-PDS dendrimer (G2, cystamine core) in phosphate buffer
(0.05 M NaH2PO4/Na2HPO4, 0.1 M NaCl, 0.01 EDTA, pH 7.4). For
example, polymer P(Hy)-SH (80 mg; 4.9 ␮mol –SH groups) was
dissolved in 1.3 mL of phosphate buffer and added into a stirring
solution of 3.2 mg of D-PDS (7.1 ␮mol PDS groups) in 1.3 mL of
phosphate buffer. After 4 h of stirring at room temperature, the
reaction mixture was diluted with 4 mL of distilled water, and LMW
impurities were removed by gel filtration (Sephadex G-25, distilled
water). The final star precursor (polymer precursor D2-SS-P(Hy))
was obtained by freeze-drying.
Star polymer precursor D2-GFLG-P(Hy) (Table 2) was prepared
by the reaction of the carboxyl group of the GFLG-OH termi-
nal sequence of semitelechelic polymer P(Hy)-COOH with amino
groups of D-NH2 dendrimer (G2, diaminobutane core) in DMF.
Polymer P(Hy)-COOH (100 mg; 6.2 ␮mol COOH groups) and HOBT
(5.78 mg, 42.7 ␮mol) were dissolved in 2.4 mL of DMF and mixed
with 6.6 ␮L of DIPC (5.4 mg, 42.7 ␮mol). After 5 min of stirring, a
2.2 mg solution of D-NH2 (G2, diaminobutan core, 9.1 ␮mol amino
groups) in 550 ␮L of DMF was added to the reaction mixture. After
20 h of stirring at room temperature, the reaction mixture was
diluted with 6 mL of methanol, and LMW impurities were removed
by gel filtration (Sephadex LH-20, solvent methanol). The star poly-
mer was isolated by precipitation into acetone.
Statistical copolymers of HPMA with Ma-ah-NHNH2 (polymer
P(Hy), Table 1) containing free hydrazide groups was prepared by
radical copolymerization in methanol (AIBN, 0.8 wt%; monomer
concentration 18 wt%; molar ratio HPMA: Ma-ah-NHNH2 93:7;
60 ◦C; 17 h) as previously described (Etrych et al., 2008b). Polymer
conjugate P(DoxH) (Table 2) bearing Dox attached via pH-sensitive
hydrazone bond was prepared by the reaction of polymer precursor
P(Hy) with Dox.HCl in methanol in the dark as described (Etrych
et al., 2001).
Statistical polymer conjugate P(DoxA) (Table 2) bearing Dox
attached via enzymatically degradable oligopeptide sequence was
prepared as described (Ulbrich et al., 2000).
2.4. Synthesis of star precursors and polymer conjugates
All the star polymer precursors/conjugates were prepared by
grafting the reactive semitelechelic HPMA copolymer precursors
onto the 1st, 2nd or 3rd generation (G1, G2 or G3) PAMAM den-
drimers containing terminal amino or PDS groups (Fig. 2.).
Polymer precursors D1-P(Hy), D2-P(Hy) and D3-P(Hy) (Table 2)
containing Boc-protected hydrazide groups were prepared by
aminolysis of TT groups of semitelechelic HPMA copolymer P(Hy)-
TT with amino groups of PAMAM dendrimers (D-NH2) (G1, G2
In polymer precursors D1-P(Hy)–D3-P(Hy), D2-SS-P(Hy) and D2-
GFLG-P(Hy), the free hydrazide groups needed for Dox attachment
to the copolymer were obtained by removing the protective Boc

532
T. Etrych et al. / European Journal of Pharmaceutical Sciences 42 (2011) 527–539
Table 2
Characteristics of the dendritic polymer precursors and the polymer–Dox conjugates.
Polymer or conjugate
Used dendrimer (type, generation)
Biodegradable spacerc
Mw (g/mol)
Mw/Mn
Rh (nm)
Dox (wt%)
P(DoxH
P(DoxA
D1-P(Hy)
D2-P(Hy)
D3-P(Hy)
D2-P(DoxH
D2-P(DoxA
D2-SS-P(Hy)
D2-SS-P(DoxH
D2-SS-P(DoxA
)
)
–
–
–
–
–
–
–
–
–
32,500
46,000
1.86
1.96
1.65
1.76
1.69
1.72
1.82
1.64
1.75
1.92
1.70
1.75
4.3
4.8
8.5
9.8a
7.4b
–
–
–
D-NH2, G1
D-NH2, G2
D-NH2, G3
D-NH2, G2
D-NH2, G2
D-PDS, G2
D-PDS, G2
D-PDS, G2
D-NH2, G2
D-NH2, G2
110,000
186,000
294,000
202,000
248,000
178,000
201,000
268,000
180,000
215,000
12.7
15.8
13.1
13.7
12.3
13.2
13.9
14.8
15.2
)
)
9.6a
10.6b
–
S-S
S-S
S-S
GFLG
GFLG
)
)
10.2a
9.8b
–
D2-GFLG-P(Hy)
D2-GFLG-P(DoxH
)
9.2a
a
Dox attached via pH-sensitive hydrazone bond.
Dox attached via amide bond to oligopeptide GFLG spacer.
GFLG sequence degradable by lysosomal enzymes or reductively degradable S-S bridge.
b
c
groups from the hydrazides with concentrated TFA. Polymer conju-
gates D2-P(DoxH), D2-SS-P(DoxH) and D2-GFLG-P(DoxH) (Table 2),
which had Dox attached via a pH-sensitive hydrazone bond, were
prepared by the reaction of precursors D2-P(Hy), D2-SS-P(Hy)
and D2-GFLG-P(Hy) with Dox.HCl in methanol in the dark. The
polymer–drug conjugates were purified from LMW impurities (Dox
or its degradation products) by gel filtration on a Sephadex LH-20
column using methanol as the eluent.
and methanol–sodium acetate buffer (80:20 vol.%) for the TSKgel
G4000SWxl column; flow-rate 0.5 mL/min.
The content of free Dox was determined by HPLC: Shi-
madzu HPLC system equipped with UV–VIS detector (Shimadzu
SPD-10AVvp). The eluent was methanol–sodium acetate buffer
(80:20 vol.%) for the TSKgel G3000SWxl column; 0.5 mL/min. Con-
tent of free Dox was calculated from the area of the corresponding
peaks at ꢀ = 488 nm.
Unreacted semitelechelic polymer precursors were removed
from the star polymer precursors and conjugates by size exclusion
chromatography using a Sephacryl S-300 column with phosphate
buffer (0.01 M NaH2PO4/Na2HPO4, 0.14 M NaCl, pH 7.4) as the elu-
ent (UV detection, 240 or 488 nm). Fractions containing HMW
polymers were collected, desalted on PD-10 columns (using water
as the eluent) and freeze-dried.
The size distribution (hydrodynamic radius (Rh)) was investi-
gated by dynamic light scattering (DLS) using ALV-6010 Multiple
tau digital correlator equipped with CGE Goniometer system,
22 mW He–Ne laser (wavelength ꢀ = 632.8 nm) and pair of
avalanche photodiodes operated in a pseudo-cross-correlation
mode. The measurements were made at a 90◦ angle. The val-
ues were means of at least five independent measurements. The
measured intensity correlation function g2(t) was analyzed using
the algorithm REPES (Jakesˇ, 1995) performing the inverse Laplace
transformation.
2.5. Characterization of polymers and polymer–Dox conjugates
The total content of Dox in polymer–Dox conjugates was
determined spectrophotometrically on Helios ␣ (Thermochrom)
spectrophotometer. Molar absorption coefficients of the free drug
(ε488 = 11,500 L mol−1 cm−1 (water)) and modified Dox (Ma-ah-
NHN = Dox: ε488 (water) = 9800 L mol−1 cm−1) (Etrych et al., 2008b)
were used for calculation of the Dox content.
The content of TT groups was determined by UV spectropho-
tometry in methanol using the molar absorption coefficient
ε305 = 8400 L mol−1 cm−1 estimated for 3,3ꢀ-disulfanediylbis[1-(2-
thioxothiazolidin-3-yl)propan-1-one].
The content of PDS groups was determined by UV spectropho-
tometry after reaction with dithiothreitol (Ulbrich et al., 2003). The
content of thiol groups was determined by reaction with Ellman’s
reagent (Ellman, 1959).
The content of hydrazide or amino groups was determined by
a modified TNBSA assay as described (Etrych et al., 2001). Molar
absorption coefficient ε500 = 17,200 L mol−1 cm−1 estimated for the
model reaction of Ma-ah-NHNH2 with TNBSA was used.
The content of GFLG sequences was determined by amino acid
analysis (Shimadzu, Japan, precolumn OPA derivatization) as pre-
viously described (Ulbrich et al., 2000).
2.6. In vitro release of Dox
The rate of Dox release from polymer–Dox conjugates (polymer
concentration equivalent to 0.5 mM Dox) was investigated in phos-
phate buffers at pH 5.0 or 7.4 (0.1 M phosphate buffer with 0.05 M
NaCl) at 37 ◦C (hydrazone conjugates) and in a solution of cathepsin
B (5 × 10−7 M) in phosphate buffer (pH 6, 0.1 M phosphate buffer
with 0.05 M NaCl, 1 M in EDTA, 3 mM in reduced glutathione) at
37 ◦C (amide conjugates) using extraction of the released Dox into
organic solvent followed by HPLC analysis as described previously
(Etrych et al., 2002) or using GPC in aqueous methanol solution as
described in Section 2.4. The activity of cysteine proteinase cathep-
sin B was determined using Bz-Arg-NAp as a substrate (Rejmanová
et al., 1983). All drug-release data are expressed as the amounts of
free drug relative to the total drug content in the conjugates. All
experiments were carried out in triplicates.
2.7. In vitro degradation of the high molecular weight star
polymer conjugates
Determination of molecular weight and polydispersity of all
prepared polymers and the content of free polymer in star polymer
precursors or conjugates were determined by GPC/HPLC Shimadzu
system equipped with UV–VIS (Shimadzu SPD-10AVvp), refrac-
tive index Optilab-rEX and multiangle light scattering DAWN EOS
detectors (both Wyatt Technology Co.). The eluent was 0.3 M
sodium acetate buffer pH 6.5 for the Superose 6 HR 10/30 column
Polymer precursor D2-GFLG-P(Hy) was incubated in sodium
phosphate buffer (0.05 M sodium phosphate, 0.1 M NaCl, 1 mM
EDTA, 3 mM GSH, pH 6) containing 0.5 ␮M cathepsin B at 37 ◦C
at a final polymer concentration of 15 mg/mL. The activity of the
cysteine proteinase cathepsin B was determined every 24 h using
Bz-Arg-NAp as the substrate (Rejmanová et al., 1983). The enzyme
concentration of cathepsin B was maintained at 5 × 10−7 M, and

T. Etrych et al. / European Journal of Pharmaceutical Sciences 42 (2011) 527–539
533
decreased enzyme activity was compensated by the addition of
3. Results and discussion
fresh stock solution of cathepsin B. In the predetermined time inter-
vals, the molecular weight of the polymers was tested using HPLC.
Polymer–Dox conjugates D2-SS-P(DoxH) and D2-SS-P(DoxA)
were incubated in sodium phosphate buffer (0.05 M sodium phos-
phate; 0.1 M NaCl; 1 mM EDTA; pH 6) containing either no GSH,
0.05 mM GSH or 3 mM GSH at 37 ◦C with a final polymer con-
centration of 15 mg/mL. In the predetermined time intervals, the
molecular weight of the samples was tested using GPC.
Degradation of polymer precursor D2-SS-P(Hy) was studied
using dynamic light scattering (DLS). Dendritic polymer precursor
D2-SS-P(Hy) was incubated in sodium phosphate buffer (0.05 M
sodium phosphate, 0.1 M NaCl, 1 mM EDTA, pH 6) containing 3 mM
GSH at 37 ◦C at a final polymer concentration of 5 mg/mL. The
solution was filtered with a 0.45 ␮m PVDF filter into a dust-free
measuring cell covered with a silicone septum. The DLS signal of the
sample was sequentially recorded for 2 h (acquisition time 40 s) to
check the stability of the system and obtain the size of the unmod-
ified, molecularly dissolved material. The measured solution was
then transferred from the measuring cell back into the vial, and the
GSH solution was added to obtain a 3 mM GSH concentration. The
mixture was quickly stirred and injected into the measuring cell
(using the same syringe and filter to prevent the loss of the volume
and material). The DLS of the solution was further recorded in the
predetermined time intervals.
In our previous papers, we have demonstrated a high efficiency
of HMW HPMA copolymer–Dox conjugates in the treatment of
tumors in mice (EL4 T-cell lymphoma). Both micellar (Chytil et al.,
2008) and graft copolymer (Etrych et al., 2008a) systems have
shown excellent in vivo anti-tumor activity, but their polydispersity
was rather high, the structures were not perfectly defined and the
reproducibility of the synthesis did not allow for the preparation of
larger batches. In this study, we focused on improving the proper-
ties of the biodegradable HMW drug delivery systems by designing
new structures of the HMW HPMA copolymer–Dox conjugates with
well-defined architecture and proper control of molecular weight
(with preservation of low polydispersity). In addition, we tried to
synthesize polymer structures that could efficiently accumulate in
solid tumors but still be eliminated after drug release. The new
systems were based on the same principles used in previous HMW
copolymer–Dox conjugates (i.e., a high molecular weight hydra-
zone or amide bond-bound Dox and a HPMA copolymer-based
structure to retain the potential for high in vivo anti-cancer activ-
ity).
The HMW polymer carrier was prepared by grafting the semit-
elechelic HPMA copolymers (which had molecular weights below
the renal threshold and bearing Dox or hydrazide groups for subse-
quent Dox attachment) onto a PAMAM dendrimer core to form the
P(Hy)-TT
CN
or P(DoxA)-TT
NH
+
y
x
O
n
O
O
D-NH2 (G1 - G3)
R
HN
HO
D1-P(Hy) (G1),
NH2
D2-P(Hy) or D2-P(DoxA) (G2)
D3-P(Hy) (G3)
P(Hy)-SH
CN
or P(DoxA)-SH
NH
+
S S
y
O
x
O
D-PDS (G2)
O
n
R
HN
HO
SS
N
D2-SS-P(Hy) or D2-SS-P(DoxA)
CN
P(Hy)-OH
DIPC
HOBT
GFLG NH
NH
+
y
x
D-NH2 (G2)
n
O
O
O
R
HN
HO
NH2
D2-GFLG-P(Hy)
for D2-SS-P(Hy); D2-GFLG-P(Hy);
Dx-P(Hy) (x = 1-3)
for D2-P(DoxA); D2-SS-P(DoxA)
GFLG NH
OH
HN
O
CH3
O
NH
R =
R =
NH
HN
O
5
O
OH
O
O
O
HO
O
HO
OH
O
Fig. 3. Scheme of the synthesis of star precursors bearing Boc-protected hydrazide groups and polymer conjugates with Dox bound by amidic bond.

534
T. Etrych et al. / European Journal of Pharmaceutical Sciences 42 (2011) 527–539
R
R
y
x
y
x
TFA
n
n
O
O
O
O
HN
HN
HO
HN
HN
HO
5
5
O
O
NH
NH2
NH
Dx-P(Hy) (x = 1-3)
D2-SS-P(Hy)
D2-GFLG-P(Hy)
HN
O
O
Dox.HCl
R
z
for D2-P(DoxH) and Dx-P(Hy) (x = 1-3)
y
x
n
O
O
O
CN
HN
HN
HN
HO
R =
NH
5
5
O
O
O
for D2-GFLG-P(DoxH) and D2-GFLG-P(Hy)
NH
NH2
NH
N
O
O
OH
OH
CN
OH
R =
GFLG NH
NH
D2-P(DoxH)
OH
D2-SS-P(DoxH)
D2-GFLG-P(DoxH)
O
O
O
for D2-SS-P(DoxH) and D2-SS-P(Hy)
NH2 .HCl
O
CN
O
NH
R =
S S
NH
OH
O
Fig. 4. Scheme of the synthesis of star polymer conjugates with Dox bound by hydrazone bond.
structures given in Figs. 3 and 4. Semitelechelic polymer precursors
were grafted onto the PAMAM dendrimer via a stable amide bond
or biodegradable linkages containing disulfide or GFLG sequences.
The grafts formed HMW star polymer carrier systems that
exceeded the size limit for glomerular filtration of HPMA-based
polymers but still allowed elimination from the organism after
degradation.
polymer–Dox conjugate prepared by Wang et al. (2000). Indeed,
both polymers contained Dox attached through a GFLG spacer
and polymer end-groups designed for aminolytic conjugation with
PAMAM dendrimers. Nevertheless, the synthetic procedure used
by Wang et al. was limited to small polymers (Mw = 5400 g/mol)
because of the use of polymer chain transfer polymerization (3-
mercaptopropionic acid used as polymer chain transfer agent).
Conversely, the use of ABIC-TT afforded full control of molecular
weight and allowed the synthesis of semitelechelic polymer pre-
cursors with molecular weights close to the renal threshold. In
addition, the reactive TT end-groups, which were less sensitive to
hydrolysis (competing with aminolysis during synthesis) than the
succinimidyl groups used by Wang et al. were directly introduced
by polymerization.
The semitelechelic copolymers bearing an end-chain thiol-
reactive PDS group (polymers P(Hy)-PDS and P(DoxA)-PDS) were
prepared by aminolysis of polymers P(Hy)-TT and P(DoxA)-TT with
PDEA. Due to the slightly lower yield of the modification reac-
tion, the functionality of polymers P(Hy)-PDS and P(DoxA)-PDS
decreased to 1.1. The molecular weight and polydispersity, how-
ever, remained almost unchanged (Table 1).
3.1. Synthesis of polymer precursors and modified dendrimers
Radical solution copolymerization of HPMA with either a
monomer bearing Boc-protected hydrazide groups (Ma-ah-NHNH-
Boc) or a monomer bearing Dox attached via an amide bond
(Ma-GFLG-Dox) was initiated with ABIC-TT. This bifunctional azo-
initiator, which contained reactive TT groups, was used for the
synthesis of polymer precursors, semitelechelic HPMA copolymers.
A previous study has shown that polymerization can be precisely
controlled by initiator and monomer concentrations and polymer-
ˇ
ization temperature (Subr and Ulbrich, 2006) and was employed
in the synthesis of copolymer P(Hy)-TT and P(DoxA)-TT (Table 1).
The polymerization conditions were chosen to keep the molecu-
lar weights of both copolymers under the renal threshold, which
was a prerequisite for subsequent elimination of the polymers from
the body. Despite the preference for a disproportionate termina-
tion reaction, the functionality (number of chain-terminating TT
functional groups per polymer chain) of the polymers was higher
than 1 (Mn/Mn,T ∼ 1.3 for both polymers). The structure of poly-
mer P(DoxA)-TT was similar to the structure of a semitelechelic
The semitelechelic copolymers bearing an end-chain thiol group
(polymers P(Hy)-SH and P(DoxA)-SH) were prepared by the reduc-
tion of PDS groups of polymers P(Hy)-PDS and P(DoxA)-PDS with
excess DTT to avoid undesired crosslinking. The molecular weight
of the polymers only slightly increased and functionality slightly
decreased, which indicated just minor amount of a side reaction
or polymer diblock formation (disulfide formation). Nevertheless,

T. Etrych et al. / European Journal of Pharmaceutical Sciences 42 (2011) 527–539
535
bonds (polymers D2-SS-P(Hy), D2-SS-P(DoxH) and D2-SS-P(DoxA))
or an enzymatically degradable oligopeptide GFLG sequence (poly-
mers D2-GFLG-P(Hy) and D2-GFLG-P(DoxH)). In all cases, the
products of the grafting reactions were high molecular weight
polymers with a star structure and a narrow distribution of molec-
ular weights (Mw = 110,000–295,000 g/mol; Mw/Mn ∼ 1.7), which
made them applicable as drug carriers for passive targeting to solid
tumors. The influence of the dendrimer structure on the molec-
ular characteristics of the conjugates was studied with 1st, 2nd
and 3rd generation D-NH2 conjugates containing 8, 16 and 32 sur-
face amino groups (polymers D1-P(Hy)–D3-P(Hy)). An increased
number of surface groups enhanced the loading capacity for the
semitelechelic copolymers, which was shown by the significant
increase in molecular weight (from 110,000 to 295,000 g/mol) and
hydrodynamic radius (Rh) (from 8.5 to 15.8 nm) of the star poly-
mers. The molecular weights indicated that an average of four to
five semitelechelic polymers were attached to the D-NH2 in poly-
mer D1-P(Hy), seven to eight in polymer D2-P(Hy) and eleven to
twelve in polymer D3-P(Hy). The ratio of modified to original amino
groups of D-NH2 strongly decreased with increasing generation of
dendrimer, which was probably due to the increased steric hin-
drance for access of HPMA copolymers to more dense amino groups
on the surface of the higher generation dendrimers. The aim of this
study was to select an appropriate dendritic polymer carrier sys-
tem, and we chose the 2nd generation of dendrimers for further
studies because they had sufficient molecular weights to achieve
effective passive tumor targeting while limiting the residual amino
groups with potential toxicity.
The polydispersity of all of the star polymers was comparable
to the semitelechelic precursors and was significantly narrower
than that found for previously studied graft and branched HMW
polymer–Dox conjugates (Etrych et al., 2008a; Ulbrich et al., 2003).
Molecular characteristics of star copolymers, which were prepared
from the 2nd generation of dendrimers, corresponded with that of
polymer D2-P(Hy). The enhanced molecular weight of copolymers
D2-P(DoxA) and D2-SS-P(DoxA) was caused by the higher molecu-
lar weight of semitelechelic polymers P(DoxA)-TT and P(DoxA)-SH.
Neither the method of synthesis (non-degradable or degradable
structure with S-S bond or GFLG sequence) nor the type of the den-
drimer (different core or surface groups) significantly influenced
the molecular weight, polydispersity or Rh of dendritic polymers
derived from 2nd generation of dendrimers.
these small changes in characteristics were not crucial and poly-
mers were used for further reactions.
The semitelechelic copolymer bearing an end-chain biodegrad-
able oligopeptide (polymer P(Hy)-COOH) was prepared by
aminolysis of polymer P(Hy)-TT with an oligopeptide H-GFLG-OH.
Similar to aminolysis with PDEA, the lower yield of the reaction
resulted in a decreased functionality of polymer P(Hy)-COOH (close
to 1) without observed changes in molecular weight and polydis-
persity.
The semitelechelic polymers P(Hy)-TT, P(DoxA)-TT and P(Hy)-
COOH terminating in TT or GFLG-OH groups were used for grafting
to the D-NH2 dendrimer. The thiol-terminated semitelechelic poly-
mers P(Hy)-SH and P(DoxA)-SH were used for grafting to the
D-PDS dendrimer. Characteristics of the semitelechelic polymers
are shown in Table 1. Their structures and scheme of synthesis are
given in Fig. 1.
Control polymer–Dox conjugates P(DoxH) and P(DoxA), which
did not have reactive end-chain groups, had similar molecular
weight and polydispersity values as their semitelechelic analogues.
A D-PDS dendrimer containing reactive PDS groups was pre-
pared in methanol by aminolysis of the active ester of SPDP with
amino groups of the D-NH2 dendrimer. The reaction mixture was
purified on a Sephadex LH-20 column (260 nm UV detection) using
methanol as the eluent. The modified dendrimer was character-
ized by the number of PDS groups (degree of substitution), and
its purity and chemical structure were determined by HPLC and
NMR spectroscopy (1H, 13C NMR and COSY), respectively. In five
independent measurements, we observed an average of 15.7 PDS
groups per molecule of dendrimer, which signified nearly complete
functionalization. The HPLC results showed a single peak (retention
time – 10.79 min).
The positions of the corresponding peaks were determined from
the 1H, 13C and COSY experiments. Due to the fact that methanol-
d6 was used as a solvent, no signals from NH and NH2 groups were
observed in the spectrum. The D-NH2 dendrimer (G2, cystamine
core) could be characterized by the five different signals in the
1H spectrum (Fig. 5A). Due to the symmetry of the structure, we
observed 16 CH2 groups at positions 1 (according to the numer-
ation of the molecular fragment, see Fig. 5A), 28 (16 + 8 + 4) CH2
groups at positions 2, 28 (16 + 8 + 4) CH2 groups at position 3, 32
(16 + 8 + 4 + 4) CH2 groups at position 4 and 12 (8 + 4) CH2 groups
at position 5. The intensity of the relevant peaks conformed to the
number of appropriate molecular groups, which was proof that the
investigated molecules matched the presented structure.
For the modified dendrimer D-PDS, attachment of PDS groups
led to the slight shift of all signals in the molecular fragment given in
Fig. 5B. This shift can be explained by the changes in interaction and
conformation of the relevant molecular segments in the presence of
PDS groups. A comparison of signal intensities from PDS groups and
dendrimer molecules led to the conclusion that all amino groups
on the dendrimers were replaced with PDS groups.
Removing the protective Boc groups from the hydrazides in
precursors D1-P(Hy)–D3-P(Hy), D2-SS-P(Hy) and D2-GFLG-P(Hy)
with TFA did not change the molecular weight or polydispersity
of the polymers. Star polymer–Dox conjugates D2-P(DoxH), D2-SS-
P(DoxH) and D2-GFLG-P(DoxH), which contained Dox bound by
a pH-sensitive hydrazone bond, were prepared by a previously
described procedure (Etrych et al., 2001) from polymer precur-
sors D2-P(Hy), D2-SS-P(Hy) and D2-GFLG-P(Hy), respectively. In all
cases, conversion was nearly complete and attachment of Dox had
no significant influence on molecular weight, polydispersity or Rh
of the polymer conjugates.
3.2. Synthesis of star polymer precursors and conjugates
The Rh of the polymer coil of the dendritic polymer conju-
gates in aqueous solution was much higher than that of linear
polymer–drug conjugates P(DoxH) and P(DoxA). Grafting of the
dendrimers with a number of semitelechelic polymers led to an
increase in Rh from about 4–5 nm to a radius that was 2- to 4-times
higher. This fulfilled the prerequisite criteria for enhanced accumu-
lation of polymers in solid tumors due to the EPR effect. Schemes
of the grafting reactions and structures of the HMW polymer pre-
cursors and conjugates are given in Figs. 3 and 4.
The star polymer conjugates were prepared by grafting semit-
elechelic polymers P(Hy)-TT, P(DoxA)-TT, P(Hy)-SH, P(DoxA)-SH
and P(Hy)-COOH onto the dendrimers containing amino (D-NH2)
or thiol-reactive PDS (D-PDS) groups. The structures of the den-
drimers used in the synthesis are shown in Fig. 2. We synthesized
three structurally different types of star polymer conjugates.
One type contained a stable amide bond between the polymer
grafts (P(Hy)-TT or P(DoxA)-TT) and central D-NH2 dendrimer and
was non-degradable (polymers D1-P(Hy)–D3-P(Hy), D2-P(DoxH)
and D2-P(DoxA)). The other two types were biodegradable poly-
mer constructs containing either reductively degradable disulfide
Non-degradable conjugate D2-P(DoxA) had structural similar-
ity with the conjugates described by Wang et al. (2000). The only
differences were that Wang et al. used significantly shorter poly-

536
T. Etrych et al. / European Journal of Pharmaceutical Sciences 42 (2011) 527–539
Fig. 5. 1H NMR spectra of PAMAM dendrimer (A) and modified dendrimer D-PDS (B).
P(DoxA), D2-P(DoxA) and D2-SS-P(DoxA) (GFLG spacer) were car-
ried out in the presence of the lysosomal enzyme cathepsin B
(5 × 10−7 M) (Fig. 6). The fastest release was observed for the lin-
ear polymer conjugate (polymer P(DoxA)); about 40% of Dox was
released within 48 h of incubation (37 ◦C) in experiment simulat-
ing conditions in lysosomes of target cells. Star HMW conjugates
D2-P(DoxA) and D2-SS-P(DoxA) showed a slower rate of release
than linear conjugate P(DoxA) because of structural differences.
Only about 15% of Dox was released with 48 h of incubation with
conjugate D2-P(DoxA), which was probably due to steric hindrance
of access of bulky enzyme to its HMW polymeric GFLG substrate.
Conjugate D2-SS-P(DoxA) had a faster release rate than conjugate
D2-P(DoxA) because it had a degradable skeleton that could be bro-
ken down by glutathione during incubation, which resulted in a
linear polymer with better substrate access for the enzyme. Wang
et al. (2000) also reported results of in vitro drug release, and they
mer grafts and higher dendrimer generation, which resulted in a
higher amount of residual amino groups and a significantly lower
rate of Dox release. Both types of conjugates were HMW and
non-biodegradable; thus, their application in human therapy is
questionable because of the high probability of undesirable poly-
mer accumulation in the body. In the current study, however, we
only used non-degradable conjugate D2-P(DoxA) as a control. For
animal treatments, we suggest the use of the reductively or enzy-
matically degradable conjugates, which have the potential to be
excreted by glomerular filtration (as discussed in Section 3.4).
3.3. In vitro drug release
A previous study has shown that the release of Dox from its
polymer conjugates was a prerequisite for its biological activity
(Duncan, 1987). Studies of in vitro Dox release from conjugates

T. Etrych et al. / European Journal of Pharmaceutical Sciences 42 (2011) 527–539
537
45
40
35
30
25
20
15
10
5
300
250
200
150
100
0
0
10
20
30
40
50
time (h)
50
0
Fig. 6. The release of Dox from linear polymer conjugate P(DoxA) and star polymer
conjugates D2-P(DoxA) and D2-SS-P(DoxA) (Dox attached via amide bond) incubated
in phosphate buffer containing cathepsin B (5 × 10−7 M) at 37 ◦C. (ꢀ —) conjugate
P(DoxA); (ꢁ —) conjugate D2-P(DoxA); (• —) conjugate D2-SS-P(DoxA); n = 3.
0
1
2
3
4
10 20 30 40 50 60 70 80
time (h)
Fig. 8. Degradation of polymers D2-SS-P(DoxH), D2-SS-P(DoxA) and D2-GFLG-P(Hy)
in phosphate buffer (pH 6) containing 3 mM GSH or 0.5 ␮M of cathepsin B at 37 ◦C.
(ꢁ —) polymer D2-SS-P(DoxH), 3 mM GSH; (• —) polymer D2-SS-P(DoxA), 3 mM GSH;
(ꢂ - - -) polymer D2-GFLG-P(Hy), 0.5 ␮M of cathepsin B.
only found that about 2% of Dox was released within 48 h of incu-
bation. It is likely that a more compact conjugate structure (as
discussed in Section 3.2) considerably enhanced steric hindrance
in the Wang et al. study.
In summary, the rate of Dox release from the polymer–drug
conjugates with a GFLG spacer was significantly dependent on the
detailed structure of the HMW star polymer carriers.
3.4. In vitro degradation of star polymer carriers
Results of pH-dependent chemical hydrolysis showed that the
hydrazone bond used for Dox attachment in both a linear (polymer
P(DoxH)) and dendritic polymer conjugates (D2-P(DoxH), D2-SS-
P(DoxH) and D2-GFLG-P(DoxH)) was fairly stable in buffer solutions
at pH 7.4 and 37 ◦C (Fig. 7), which model the blood environment.
Only a slight release of Dox, up to 7% of Dox, was observed within
24 h of incubation for all of them. By contrast, around 90% of Dox
was released in the same period of time from the conjugates after
incubation in a buffer of pH 5 at 37 ◦C, which simulated conditions
in endosomes of target cells. In contrast to enzymatic hydrolysis,
the effect of size and structure of the polymer–drug conjugates was
not observed in this case. In addition, the effect of steric hindrance
to hydrolysis was negligible in this case.
Previously, we have shown that passive targeting of the HPMA-
based copolymers to solid tumors via the EPR effect (Etrych
et al., 2008a; Noguchi et al., 1998; Pimm et al., 1996) strongly
depends on the molecular weight of the polymer (i.e., the HMW
polymers accumulated most effectively). Linear HPMA copoly-
mers are non-biodegradable polymers with limited potential to
be removed from an organism by glomerular filtration when
molecular weight exceeds the renal threshold (approximately
50,000 g/mol). The new biodegradable star HMW HPMA-based
copolymers (Mw ∼ 110,000–294,000 g/mol) consisted of polymer
precursors (Mw ∼ 30,000 g/mol for polymers P(Hy)-SH and P(Hy)-
COOH or 43,500 g/mol for polymer P(DoxA)-SH) grafted onto small
rigid dendrimer cores via biodegradable oligopeptide or disulfide
bridges.
The degradation of the conjugates was also studied in phos-
phate buffer (pH 6) containing either cathepsin B or GSH to
simulate the intracellular environment (lysosomes, cytoplasm).
Conjugates D2-SS-P(DoxH) and D2-SS-P(DoxA), which contained
disulfide spacers susceptible to reductive degradation, were
degraded rapidly in incubation media containing 3 mM GSH, which
modeled the cytosolic environment (Meister, 1991; Saito et al.,
2003). Within 4 h of incubation, both conjugates were completely
degraded to products with molecular weights below the renal
threshold (i.e., polymer fragments with molecular weights about
30,000–40,000 g/mol) (Fig. 8). The rate of degradation was com-
parable with values observed recently for other HMW polymer
conjugates (Etrych et al., 2008a).
Star polymer carrier D2-GFLG-P(Hy), which contained enzy-
matically degradable GFLG sequences, was hydrolyzed relatively
slowly, but it was almost completely hydrolyzed in the buffer
containing 0.5 ␮M cathepsin B after 72-h incubation (Fig. 8). As
expected, the hydrolysis products were polymers with molecular
weights comparable with HPMA copolymers used for the synthesis
(∼45,000 g/mol). Enzymatic degradation of polymer D2-GFLG-
P(Hy) proceeded slower than previously reported graft systems
(Etrych et al., 2008a). This finding likely resulted from steric hin-
drances, which were created by the higher molecular weight and
compact structure of polymer D2-GFLG-P(Hy) and prevented easy
enzyme access.
To resume drug release studies, the new star polymer–Dox con-
jugates complied with basic requirements for efficient anticancer
prodrugs: (i) exhibit stability under conditions modeling blood
circulation and (ii) release an active drug in an environment mim-
icking conditions in tumor cells or tissues.
100
90
80
70
60
50
40
30
20
10
0
0
5
10
15
20
25
time (h)
Fig. 7. The release of Dox from linear polymer conjugate P(DoxH) and star polymer
conjugates D2-P(DoxH), D2-SS-P(DoxH) and D2-GFLG-P(DoxH) (Dox is attached via
a hydrazone bond) incubated in phosphate buffers at 37 ◦C. (ꢁ —) Conjugate D2-
P(DoxH), pH 5; (• —) conjugate D2-SS-P(DoxH), pH 5; (ꢂ - - -) conjugate D2-GFLG-
P(DoxH), pH 5; (ꢀ —) conjugate P(DoxH), pH 5; (ꢁ- - - -) conjugate D2-P(DoxH), pH
7.4; (• - - -) conjugate D2-SS-P(DoxH), pH 7.4; (ꢂ - - -) conjugate D2-GFLG-P(DoxH),
pH 7.4; (ꢀ - - -) conjugate P(DoxH), pH 7.4; n = 3.

538
T. Etrych et al. / European Journal of Pharmaceutical Sciences 42 (2011) 527–539
Fig. 9. Graph of DLS measurements of polymer precursor D2-SS-P(Hy) before and after GSH addition (3 mM) (phosphate buffer of pH 6 at 37 ◦C).
The stability of the star polymer conjugates in buffers mim-
intratumoral environments. Controlled synthesis resulted in con-
jugates with well-defined structure, a broad range of molecular
weights and low polydispersity. A high molecular weight prede-
termined accumulation and drug delivery of the conjugates into
solid tumors due to the EPR effect. Controlled Dox release was
achieved by using either a hydrazone bond-containing spacer,
which predetermined the conjugate to pH-controlled intratumoral
or intracellular hydrolysis and drug release, or a lysosomotropi-
cally degradable GFLG oligopeptide predetermining the conjugate
to specific intracellular drug release. The degradability of the car-
rier was ensured by using reductively or enzymatically degradable
spacers with high potential for intracellular degradation. Chemical
hydrolysis of the hydrazone bond and Dox release, as well as the
reductive degradation of the carrier structure, were not dependent
on the molecular weight of the carrier or its architecture. Specific
enzymatic degradation of the lysosomotropic GFLG spacer, how-
ever, was significantly influenced by molecular weight and detailed
structure of the carrier because of steric hindrances that limited the
access of the enzyme to its macromolecular substrate. The well-
defined structure, controlled molecular weight and proper drug
release and degradation profiles suggest that the polymer-grafted
dendrimer conjugates have the potential to be efficient anti-cancer
therapies. In vitro and in vivo evaluation of toxicity and anticancer
activity of the conjugates is underway, and preliminary results have
shown excellent anti-cancer activity. Thus, a detailed study of the
biological properties of the star conjugates will be the subject of a
separate paper.
icking blood circulation was tested by incubation in a phosphate
buffer (pH 7.4), in a similar buffer containing a low concentration
of glutathione (0.06 mM) (Meister, 1991) or in human plasma. No
changes in molecular weight or polydispersity of the conjugates
were observed.
Degradation of star polymer D2-SS-P(Hy), which contained
reductively degradable disulfide bonds, was studied using DLS. It
is shown in Fig. 9 that there was no evidence of degradation dur-
ing the 2-h incubation in phosphate buffer prior to the addition of
GSH. After the addition of GSH, a reductive degradation began and
a significant change in Rh was observed. During the first 30 min, the
Rh of the polymer coils decreased from 12.3 nm to 5.3 nm, and the
reduction was nearly complete. Longer incubations did not show
significant progress in the reductive degradation and final Rh of the
polymer degradation products (the Rh was 4.8 nm).
The results of drug carrier degradation based on star HPMA
polymers demonstrated that all of the conjugates were stable
under conditions modeling the blood stream. They under-
went degradation, however, after incubation in intracellular
environment-mimicking media. Thus, the new HMW polymer–Dox
conjugates derived from dendrimers and HPMA polymers are
expected to release Dox and degrade inside tumor cells where they
will form polymer fragments that are capable of being excreted
from the body by glomerular filtration.
4. Conclusion
The present study described the synthesis, physicochemi-
cal characterization, drug release and biodegradation of new
biodegradable, high molecular weight doxorubicin conjugates
based on star HPMA polymer carriers, which were designed for
solid tumor treatment. The conjugates were synthesized by graft-
ing the reactive semitelechelic HPMA copolymer precursors onto
1st, 2nd or 3rd generation PAMAM dendrimers. The conjugates
differed in detailed structure of the spacers between Dox and
HPMA copolymer as well as the polymer grafts and the dendrimer
core, which enabled controlled drug release and degradation of
the carrier after incubation in solutions modeling intracellular or
Acknowledgements
This work was supported by the Grant Agency of the Academy
of Sciences of the Czech Republic (grants no. IAA400500806 and
No. IAAX00500803) and by the Grant Agency of the Czech Republic
(grant no. 203/08/0543).
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