HPMA copolymer-doxorubicin conjugates: The effects of molecular weight and architecture on biodistribution and in vivo activity

Article abstract of DOI:10.1016/j.jconrel.2012.06.029

The molecular weight and molecular architecture of soluble polymer drug carriers significantly influence the biodistribution and anti-tumour activities of their doxorubicin (DOX) conjugates in tumour-bearing mice. Biodistribution of N-(2-hydroxypropyl)methacrylamide (HPMA) copolymer-DOX conjugates of linear and star architectures were compared in EL4 T-cell lymphoma-bearing mice. Biodistribution, including tumour accumulation, and anti-tumour activity of the conjugates strongly depended on conjugate molecular weight (MW), polydispersity, hydrodynamic radius (R_h) and molecular architecture. With increasing MW, renal clearance decreased, and the conjugates displayed extended blood circulation and enhanced tumour accumulation. The linear conjugates with flexible polymer chains were eliminated by kidney clearance more quickly than the highly branched star conjugates with comparable MWs. Interestingly, the data suggested different mechanisms of renal filtration for star and linear conjugates. Only star conjugates with MWs below 50,000 g.mo ^-1 were removed by kidney filtration, while linear polymer conjugates with MWs near 70,000 g.mol^{- 1}, exceeding the generally accepted limit for renal elimination, were detected in the urine 36-96 h after injection. Additionally, survival of tumour-bearing mice was strongly dependent on molecular weight and polymer conjugate architecture. Treatment of mice with the lower MW conjugate at a dose of 10 mg DOX eq./kg resulted in 12% long-term surviving animals, while treatment with the corresponding star conjugate enabled 75% survival of animals.

Full text of DOI:10.1016/j.jconrel.2012.06.029

Journal of Controlled Release 164 (2012) 346–354
Contents lists available at SciVerse ScienceDirect
Journal of Controlled Release
journal homepage: www.elsevier.com/locate/jconrel
HPMA copolymer-doxorubicin conjugates: The effects of molecular weight and
architecture on biodistribution and in vivo activity
a
b
b
a,
Tomáš Etrych a, Vladimír Šubr a, , Jiří Strohalm , Milada Šírová , Blanka Říhová , Karel Ulbrich
⁎
⁎
a
Institute of Macromolecular Chemistry, Academy of Sciences of the Czech Republic v.v.i., Heyrovský Sq. 2, 162 06 Prague 6, Czech Republic
Institute of Microbiology, Academy of Sciences of the Czech Republic v.v.i., Vídeňská 1083, 142 20 Prague 4, Czech Republic
b
a r t i c l e i n f o
a b s t r a c t
Article history:
The molecular weight and molecular architecture of soluble polymer drug carriers significantly influence the
biodistribution and anti‐tumour activities of their doxorubicin (DOX) conjugates in tumour-bearing mice.
Biodistribution of N-(2-hydroxypropyl)methacrylamide (HPMA) copolymer-DOX conjugates of linear and
star architectures were compared in EL4 T-cell lymphoma-bearing mice. Biodistribution, including tumour
accumulation, and anti‐tumour activity of the conjugates strongly depended on conjugate molecular weight
(MW), polydispersity, hydrodynamic radius (Rh) and molecular architecture. With increasing MW, renal
clearance decreased, and the conjugates displayed extended blood circulation and enhanced tumour accu-
mulation. The linear conjugates with flexible polymer chains were eliminated by kidney clearance more
quickly than the highly branched star conjugates with comparable MWs. Interestingly, the data suggested
different mechanisms of renal filtration for star and linear conjugates. Only star conjugates with MWs
below 50,000 g.mo−1 were removed by kidney filtration, while linear polymer conjugates with MWs near
70,000 g.mol−1, exceeding the generally accepted limit for renal elimination, were detected in the urine
36–96 h after injection. Additionally, survival of tumour-bearing mice was strongly dependent on molecular
weight and polymer conjugate architecture. Treatment of mice with the lower MW conjugate at a dose of
10 mg DOX eq./kg resulted in 12% long-term surviving animals, while treatment with the corresponding
star conjugate enabled 75% survival of animals.
Received 21 March 2012
Accepted 21 June 2012
Available online 30 June 2012
Keywords:
Drug delivery
Tumour accumulation
Body distribution
HPMA copolymers
Pharmacokinetics
Renal elimination
© 2012 Elsevier B.V. All rights reserved.
1. Introduction
The Enhanced Permeability and Retention Effect (EPR) described by
Maeda and Matsumura is based on the preferential accumulation of
Contemporary oncology is a fast-developing branch of medicine
that exploits research achievements in other disciplines such as cell
biology, immunology, and genetics. Additionally, polymer chemistry
offers powerful tools for the development of new generations of
drugs. Novel polymer drug delivery systems (DDS) have been devel-
oped and tested in numerous preclinical and clinical studies. The
design of new polymer-drug conjugates is based on the idea that
synthetic polymer carriers [1–3] can enable passive and active
tumour-specific drug delivery. Conjugation of a chemotherapeutic
agent to a hydrophilic polymer carrier increases the solubility of
water-insoluble drugs, prolongs the circulation of a polymer-drug
conjugate in the blood, influences drug pharmacokinetics and bio-
distribution, decreases side-toxicity and induces therapy-dependent
cancer resistance as it was evidenced for HPMA copolymers of various
structures earlier [4,5].
high-molecular weight (HMW) polymer drug conjugates in solid tu-
mours, and the efficiency of this accumulation is molecular weight-
dependent [6–8]. Furthermore, fluid-phase and receptor-mediated pi-
nocytosis are the most important mechanisms underlining subsequent
uptake of water-soluble polymer drug conjugates in target tumour cells.
The balance between elimination of polymeric drugs from the
bloodstream by the kidneys, liver and other organs and extravasa-
tion from the blood vasculature into the tumour affect the effective-
ness of drug delivery [9]. The structure of a glomerular capillary wall
and the glomerular permeability of macromolecules have been de-
scribed in several papers and reviews [10–15]. Macromolecular
clearance decreases with increasing hydrodynamic radius (Rh) and
molecular weight and is influenced by polymer charge. The clearance
of negatively charged macromolecules is restricted, while the clear-
ance of positively charged macromolecules with the same molecular
weight is enhanced [10]. The renal clearance of linear flexible macro-
molecules, such as dextran and poly(vinylpyrrolidone), is up to 10
times greater than that of proteins with equivalent hydrodynamic
radii [11]. Macromolecular branching leads to decreased renal clear-
ance and significantly prolongs blood circulation time [9,16].
⁎
Corresponding authors. Tel.: +420 296 809 389; fax: +420 296 809 410.
E-mail addresses: subr@imc.cas.cz (V. Šubr), ulbrich@imc.cas.cz (K. Ulbrich).
0168-3659/$ – see front matter © 2012 Elsevier B.V. All rights reserved.
doi:10.1016/j.jconrel.2012.06.029

T. Etrych et al. / Journal of Controlled Release 164 (2012) 346–354
347
Macromolecules are typically eliminated from the body through a
2.2. Mice
kidney glomerulus, which in mice or rats contains pores approximately
4 nm by 14 nm in size [17] and 8 nm in humans [18]. Macromolecules
with a coil diameter smaller than glomerular pores permeate these
pores and are excreted from the body in the urine [11]. The threshold
for renal filtration of macromolecules with hydrodynamic radii (Rhs)
below 5 nm corresponds roughly with their molecular weights, ranging
from 30,000 to 50,000 g.mol−1, and is influenced by their molecular
architecture [9,13,19]. Noguchi demonstrated that radiolabeled poly
[N-(2-hydroxypropyl)methacrylamide)] with different molecular
weights, ranging from 4500 to 800,000 g.mol−1, accumulated in tu-
mour tissue at approximately the same rate; however, the higher mo-
lecular weight polymers accumulate in tumour tissue more than in
healthy tissue and are unable to diffuse either tissue through the vascu-
lature as rapidly as the lower molecular weight polymers [20]. The total
body distribution of 131I radiolabeled HPMA copolymers was monitored
scintigraphically for 7 days after i.v. injection into Copenhagen rats
bearing Dunning prostate carcinoma. The kinetic of tumour accumula-
tion was dependent on copolymer molecular weight. The HPMA copol-
ymers with MWs above the kidney threshold displayed continuous and
higher accumulation than polymers with shorter chains [21,22].
Sadekar et al. compared the biodistribution of 125I radiolabeled linear
HPMA copolymers with branched poly(amido amine) dendrimers con-
taining surface hydroxyl groups in ovarian-tumour-bearing mice
(A2780). They found that polymer architecture affects renal and hepatic
uptake, with dendrimers showing more persistent accumulation than
linear HPMA copolymers. This study also showed that prolonged tumour
retention may be obtained for polymers with hydrodynamic radii ap-
proximately 4 nm or higher [19].
Ideally, a drug covalently bound to a polymer conjugate should be in-
active during transport through the body and should be activated
(released) from the carrier at the site of its desired pharmacological
effect, for example, in the solid tumour or in tumour cells either by
enzymolysis or pH-controlled hydrolysis [23,24]. A broad range of
HPMA copolymer (pHPMA) based conjugates have been synthesised
and studied [25,26]. Further development has led to clinical trials for
some of these specific drug-polymer conjugates [27–30].
Nevertheless, to properly design polymer-drug conjugates with
high potential to succeed in clinical evaluation, more detailed in
vivo studies are necessary. Recently, new nanomedicines based on
biodegradable high-molecular weight (HMW) polymer carriers
designed for treatment of solid tumours have been developed
[31–33]. Among them, HMW conjugates of a star structure showed
superior properties in vivo [33]. The aim of this work is to study
the impact of the polymer carrier's MW and architecture on drug
biodistribution and in vivo anti-cancer activity of the conjugate. For
this study, HPMA copolymer conjugates bearing DOX bound by an
enzymatically degradable oligopeptide GFLG spacer were selected
because of their satisfactory stability in blood and efficient intracel-
lular drug release [34,35].
C57BL/6 (B/6) mice were obtained from the breeding colony of the
Institute of Physiology, AS CR, v.v.i. (Prague, Czech Republic). Mice
were used at 8–12 weeks of age, housed in accordance with approved
guidelines and provided food and water ad libitum. The Animal Wel-
fare Committee of the Institute of Microbiology AS CR, v.v.i., approved
all experiments.
2.3. Synthesis of monomers
N-(2-Hydroxypropyl)methacrylamide (HPMA) was synthesised
by acylating 1-aminopropan-2-ol with methacryloyl chloride in dic-
hloromethane in the presence of sodium carbonate as previously de-
scribed [36]. M.p. 69–70 °C; elemental analysis: calc., C 58.72%, H
9.15%, N 9.78%; found, C 58.98%, H 9.18%, N 9.82%.
3-(N-Methacryloylglycylglycyl)thiazolidine-2-thione (Ma-GG-TT)
was prepared by reacting N-methacryloylated glycylglycine with
4,5-dihydrothiazole-2-thiol in the presence of DCC and a catalytic
amount of DMAP [37].
N-Methacryloylglycyl-DL-phenylalanyl-L-leucylglycine
4-nitrophenyl ester (Ma-GFLG-ONp) was prepared using a previously
described procedure [38].
3-(N-Methacryloylglycyl-DL-phenylalanylleucylglycyl)
thiazolidine-2-thione (Ma-GFLG-TT) was prepared according to a lit-
erature procedure [37]. Yield: 0.954 g (39%). The purity determined
by HPLC was >99.2%; 1H NMR [(CD3)2SO]: δ 0.84 (d, 3H, CH3-Leu),
0.89 (d, 3H, CH3-Leu), 1.30–1.70 (m, 3H, CHCH2-Leu), 1.84 (s, 3H,
CH3), 2.70–3.10 (m, 2H, β-Phe), 3.45 (t, 2H, CH2S), 3.50–3.75
(m, 2H, Gly), 4.15–4.35 (m, 1H, α-Leu), 4.45–4.75 (m, 5H, CH2N,
Gly, α-Phe), 5.35 (s, 1H, CH2_), 5.69 (s, 1H, CH2_), 7.20 (m, 5H,
ArH), 8.01 (d, 1H, NH-Phe), 8.10 (m, 2H, NH-Gly), 8.25 (t, 1H,
NH-Leu).
(N-Methacryloylglycyl-DL-phenylalanylleucylglycyl)doxorubicin
(Ma–GFLG–DOX) was prepared by reacting an equivalent amount of
Ma-GFLG-ONp with DOX.HCl in DMF at 4 °C as previously reported
[36]. TLC: two spots at Rf =0.46 and 0.4, corresponding to the D-Phe
and L-Phe isomers (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 purities of all monomers mentioned above were examined by
HPLC (Shimadzu, Japan) using a reverse-phase column (Chromolith Per-
formance RP-18e 100–4.6 mm) with UV detection (230 nm, 305 nm or
488 nm), a water/acetonitrile gradient elution (0–100 vol.%), and a flow
rate of 0.5 mL/min.
2.4. Synthesis of polymer precursors and conjugates
Multivalent copolymers of HPMA with Ma-GFLG-TT (polymers 1
and 2, Table 1) were prepared by radical solution polymerisation in
DMSO at 50 or 60 °C for 6 h (AIBN, 1 or 2 wt.%; monomer concentra-
tion, 15 or 12.5 wt.%; molar ratio of HPMA: Ma-GFLG-TT, 95:5). The co-
polymers were isolated by precipitation in acetone/diethyl ether (1:3)
and purified by re-precipitation into the same mixture. Yield: 75%.
The multivalent copolymer of HPMA with Ma-GG-TT (polymer 3,
Table 1) was prepared accordingly in DMSO at 50 °C (AIBN, 1.5 wt.%;
monomer concentration, 15 wt.%; molar ratio of HPMA: Ma-GG-TT,
90:10; 7 h). Yield: 72%.
Liner polymer conjugates containing amide-bonded doxorubicin
(polymers 4–6 and 12) were prepared by aminolysis of TT groups of
polymer precursors with DOX.HCl in DMSO in presence of TEA as previ-
ously described [36]. The drug-containing polymer was isolated by pre-
cipitation in acetone/diethyl ether, purified and fractionated by single
(polymer 6) or multiple (polymer 4 and 5) gel permeation chromatog-
raphy in water (Sephacryl S-300, column 1.5×60 cm). The highest and
2. Materials and methods
2.1. Chemicals
1-Aminopropan-2-ol,
methacryloyl
chloride,
2,2′-azobis
(isobutyronitrile) (AIBN), N,N-dimethylformamide (DMF), N,N′-
dicyclohexylcarbodiimide (DCC), 3-mercaptopropionic acid, dimethy-
laminopyridine (DMAP), N-hydroxybenzotrizole (HoBT), L-leucylglycine,
glycyl-L-phenylalanine, glycylglycine, 4,5-dihydrothiazole-2-thiol, o-
phthalaldehyde (OPA), N-ethyldiisopropylamine (EDPA), dimethyl
sulfoxide (DMSO) and triethylamine (TEA) were purchased from
Sigma-Aldrich, Czech Republic. Doxorubicin hydrochloride (DOX.HCl)
was purchased from Meiji Seiko, Japan. Poly(amido amine) (PAMAM)
dendrimers were purchased from Dendritic Nanotechnologies, Inc.,
Mount Pleasant, MI, USA.

348
T. Etrych et al. / Journal of Controlled Release 164 (2012) 346–354
spectrophotometer (ε305=10,700 L.mol−1.cm−1
; methanol) [37].
Table 1
Characteristics of polymer precursors and conjugates.
The hydrodynamic radius (Rh) was determined by dynamic light scat-
tering (DLS) using an aqueous solution of polymer conjugates and a
scattering angle of 173° on a Nano-ZS, Model ZEN3600 (Malvern, UK)
zetasizer.
The content of end carboxylic groups in semitelechelic polymer
precursor 7 was determined by titration with 0.05 N NaOH using an
automatic titrator TIM900 (Radiometer). From the concentration of
Polymer
Spacer
Mw (g.mol−1
)
Mw/Mn
TT content
(mol%)
DOX
(wt.%)
Rh
(nm)
1
2
3
4
5
6
GFLG
GFLG
GG
GFLG
GFLG
GG
GFLG
GFLG
GFLG
GFLG
AcapNHN
GFLG
24,400
121,000
87,800
35,800
96,900
89,000
18,400
66,000
94,000
248,000
202,000
130,000
1.95
1.72
1.87
1.20
1.29
1.80
1.69
1.45
1.25
1.82
1.72
1.50
3.9
4.2
9.95
–
–
–
–
–
–
–
8.6
6.8
7.5
9.7
9.5
9.6
10.6
9.6
7.4
4.6
7.8
7.4
–
–
the end carboxylic groups, the average molecular weight (Mn,COOH
of semitelechelic polymer precursor 7 was calculated.
)
–
7a
8b
9b
10b
11b
12
6.9
7.9
13.7
13.1
13.8
2.6. Blood clearance and determination of DOX content in the urine, liver
and tumour tissues
C57BL/6 (B/6) male mice were inoculated subcutaneously with
1×105 EL4 lymphoma cells. When the tumour reached a volume of
50−75 mm3, the mice were injected intravenously (i.v.) with either
DOX.HCl (5 mg/kg) or conjugates 4–6 or 8–9 (15 mg DOX eq./kg repre-
sents an equitoxic dose). Blood, urine, liver and tumour tissue samples
were taken at the following times after injection: 1, 3, 6, 9, 12, 24, 30,
36, 48, and 72 h for blood and urine (5 mice per group) and 6, 12, 24,
48, and 72 h for the liver and tumour tissues (3 mice per group). The
blood samples were collected in heparinised tubes, while the liver and tu-
mour samples were excised, weighed and homogenised. The samples
were tested for their total DOX content (i.e., the sum of free and
polymer-bound DOX) and molecular weights and polydispersities of the
polymer conjugates eliminated from the body in urine.
The total DOX content in the samples was determined after quantita-
tive acid hydrolysis (1 M HCl, 1 h at 50 °C) of the respective samples. The
doxorubicinone, formed from free and polymer-bound doxorubicin after
hydrolysis, was extracted with chloroform, and the organic phase was
evaporated to dryness. The remaining solid was completely dissolved in
methanol and analysed using a gradient-based HPLC Shimadzu system
a
Semitelechelic polymer precursor.
Star polymer conjugate.
b
lowest MW fractions of each polymer were removed during the separa-
tion. The final polymer conjugates (4–6) were freeze-dried.
Semitelechelic polymer precursor 7 was prepared by precipitation
chain-transfer radical copolymerisation of 127.7 mg HPMA with
27.2 mg Ma–GFLG–DOX in 1.25 mL acetone using 1.5 mg AIBN as initi-
ator and 2.43 mg 3-mercaptopropionic acid as a chain-transfer agent.
The polymerisation was performed in a sealed ampoule under nitrogen
at 50 °C for 23 h. The polymer was removed by filtration and purified by
re-precipitation from methanol into acetone/diethyl ether (3:1). Star
polymer conjugates 8 and 9 were prepared by grafting the semi-
telechelic polymer 7 onto the amino groups of the PAMAM dendrimer.
Example of the reaction: polymer precursor 7 (33 mg) was activated by
2 mg HoBT and 3 mg DCC in 0.6 mL DMF at 15 °C. After stirring for 4 h,
the polymer was added to a solution containing 1 mg PAMAM dendri-
mer (generation G2, 16 amino groups), the reaction was left to stir gent-
ly for another 20 h, and the star polymer conjugate was isolated by
precipitation in a mixture of dry acetone/diethyl ether (1:1). After dis-
solution in phosphate-buffered saline, the conjugate was fractionated
by single gel permeation chromatography (Sephacryl S-300, column
1.5×60 cm). The highest and lowest MW fractions were removed,
and the final star polymer 9 was obtained after desalting using a
Sephadex G-25 column (distilled water eluent) and freeze-drying.
Star polymer conjugates 10 and 11 with DOX bound via a GFLG spacer
and amide bond (10) or via a hydrazone bond (11) were synthesised
accordingly by grafting PAMAM dendrimers with semitelechelic poly-
mer precursors using a procedure described in [39]. Conjugate 11,
which displays pH-controlled drug activation, was used as a control in
an in vivo study in comparison to conjugates displaying enzyme-
triggered drug release.
equipped with a Shimadzu RF-10Axl fluorescence detector (λexc
=
488 nm, λem=560 nm) [40]. Calibration was performed by injection of
exact amounts of free DOX.HCl into the blood, urine and tumour tissue
obtained from untreated animals followed by analysis (samples were
homogenised, hydrolysed and extracted) as described above. The molec-
ular weight, polydispersity and amount of polymer conjugate in each
urine sample were determined on HPLC Shimadzu system equipped
with a TSKgel G4000SW column (dn/dc=0.170 mL.g−1) and LS, RI and
fluorescence detectors after desalting the samples on PD10 columns
(700 μL urine was injected onto PD-10 column, eluent distilled water)
and freeze-drying of eluted polymer fraction. Extraction of urine samples
did not show presence of significant amount of free DOX.
2.7. In vivo evaluation of anti-cancer activity
B/6 males were subcutaneously (s.c.) transplanted with 1×105 EL4 T
cell lymphoma cells on the right flank (day 0). The mice that developed
palpable tumours reaching 50–75 mm3 were injected i.v. with 200 μL of
polymer conjugates or free drug PBS solutions (for dosage, see Results
and discussion). The doses referred to hereafter are expressed as mg of
DOX equivalent per kg. Control mice were transplanted with tumour
cells and left untreated. The animals (8 mice per group) were observed
for signs of tumour progression and acute toxicity. Tumour size, body
weight, survival time and number of long-term survivors were deter-
mined. Those surviving at least 60 days without any signs of tumour are
considered long-term survivors (LTS).
2.5. Purification and characterisation of polymers and polymer conjugates
Polymers 4–6, 8–9 and 12 were tested for free DOX content using
HPLC (Shimadzu, Superose™ 6 or TSKgel G4000SW columns). The
total DOX content was determined spectrophotometrically on a Helios
α (Thermochrom) spectrophotometer. The molar absorption coeffi-
cient in methanol (ε488=8 100 L.mol−1.cm−1) was used for the calcu-
lation of drug content.
MW and polydispersity determination of the conjugates was per-
formed on a HPLC Shimadzu system equipped with UV, an Optilab®
rEX differential refractometer and MALS DAWN® 8™ (Wyatt Technolo-
gy, USA) detectors. For these experiments, a 0.3 M acetate buffer (pH 6.5;
0.5 g/L NaN3) and a Superose™ 6 column or a 20% 0.3 M acetate/80%
methanol (v/v) buffer and TSKgel G4000SW column were used.
The content of thiazolidine-2-thione (TT) groups was deter-
2.8. Statistics
The statistical significance (Pb0.05) of the differences between vol-
umes of tumours in the various groups was assessed by applying a
two-sided Student's t-test. For each approach, three independent
experiments were conducted and differences between exposed and
mined spectrophotometrically on
a Helios α (Thermochrom)

T. Etrych et al. / Journal of Controlled Release 164 (2012) 346–354
349
control animals with an error probability of Pb0.05 were considered to
be statistically significant.
A
CH3
CH3
3. Results and discussion
x
y
Recently, increased attention has been paid to the development of
new HMW polymer drug carrier systems that will enable enhanced
tumour accumulation via the EPR effect [33,41]. We have previously
demonstrated the remarkable in vivo efficiencies of HMW HPMA
copolymer-DOX conjugates designed for solid tumour treatment. Spe-
cifically, we found that increasing the molecular weight of such polymer
carriers above the limit of renal filtration led to decreased elimination of
the polymers from circulation by kidney clearance, which resulted in
prolonged blood circulation half-times in the organism and enhanced
accumulation of the drug in solid tumours due to the EPR effect. To
avoid undesirable long-term accumulation of the HMW polymer carrier
in the body, such polymer constructs must contain biodegradable
spacers susceptible to body-related biodegradation, namely hydrolysis
or reduction [33,42]. Polymer carriers based on HPMA may either be
eliminated from the organism by the quick process of renal filtration
or by slower processes via the liver and bile.
This paper is focused on the synthesis of HMW polymer-DOX conju-
gates differing in molecular weight, polydispersity and molecular archi-
tecture and the study of their physicochemical and preliminary
biological properties, specifically tumour and liver accumulation, blood
clearance and urine elimination. Four of the synthesised conjugates
were linear polymers (see Fig. 1) that differed in polydispersity and mo-
lecular weight, and their molecular weights were either below or above
the generally accepted kidney elimination threshold (~40,000 g.mol−1).
The other synthesised polymer-DOX conjugates were prepared by
grafting semitelechelic HPMA copolymers onto a PAMAM dendrimer
core to form HMW star structures (see Fig. 2). Except for conjugate 11,
all synthesised polymer conjugates contained a GFLG spacer and an
amide bond-bound DOX.
O
O
O
HN
HN
HN
O
O
HO
NH
CH3
CH3
NH CH3
O
HN
O
OH
CH3
O
O
OH
OCH3
HO
HO
O
OH
O
B
The primary goal of this study was to demonstrate that molecular
architecture, polydispersity and molecular weight strongly influence tu-
mour accumulation, blood clearance and urine elimination of
polymer-DOX conjugates with direct impact on their anti-cancer activi-
ties. Moreover, we aimed to show that water-soluble linear polymers,
even with molecular weights above the generally accepted renal thresh-
old for HPMA polymers (~40,000 g.mol−1), could also be removed by
renal filtration.
CH3
CH3
x
y
O
O
HN
HN
O
HO
NH
CH3
3.1. Synthesis of polymer precursors and conjugates
O
O
Multivalent polymer precursors 1 to 3 with molecular weights
ranging from 25,000 to 120,000 g.mol−1 and containing reactive TT
groups susceptible to aminolysis were prepared by radical solution
copolymerisation in DMSO [37]. These precursors enabled synthesis
of linear polymer-DOX conjugates 4–6 and 12 (Table 1) by simple
aminolytic reaction with DOX.HCl in DMSO containing TEA as a
base. Polymer conjugates 4 and 5 were subsequently fractionated by
gel chromatography to obtain polymer-DOX conjugates with low
polydispersities and with MWs below (conjugate 4) and above (con-
jugates 5 and 12) the renal threshold [43].
HN
O
OH
CH3
OH
O
OCH3
HO
Semitelechelic polymer 7 (Table 1) was prepared by radical solution
copolymerisation of HPMA with Ma–GFLG–DOX initiated with AIBN in
the presence of 3-mercaptopropionic acid as the chain-transfer agent.
HO
O
OH
O
Use of this method enabled preparation of
a semitelechelic
Fig. 1. Structures of polymer-DOX conjugates: (A) conjugates
4
or
5
containing
polymer-DOX precursor with a functionality (or number of end chain
carboxylic groups per polymer chain) close to 1 (Mn/Mn,COOH ~0.97).
Star polymer conjugates 8 and 9, which differ only in molecular
weight, were prepared by grafting semitelechelic polymer 7 onto a
PAMAM dendrimer (generation 2) containing 16 amino groups
using a DCC coupling method. The products of the grafting reaction
were water-soluble HMW polymers with star structures. Polymer
amide-bonded DOX attached to a GFLG spacer, which is degradable by lysosomal en-
zymes, (B) conjugate 6 containing amide-bonded DOX attached to a nondegradable
GG spacer.
conjugates 8 and 9 were fractionated by gel chromatography to
obtain polymer conjugates with low polydispersities, comparable to
the polydispersities of polymer conjugates 4 and 5. The molecular

350
T. Etrych et al. / Journal of Controlled Release 164 (2012) 346–354
the structure of the oligopeptide spacer and the concentration of the re-
S
OH
spective enzyme responsible for its cleavage [23,33,44]. No significant
DOX release from polymer conjugates containing a diglycine spacer was
observed during incubation in human serum [34] or lysosomal enzymes.
Such a spacer is considered nondegradable (here, conjugate 6) because
the conjugate does not release drug, and thus, this spacer was used in
this study. Conversely, conjugates containing a GFLG spacer release
DOX in the presence of cathepsin B (5×10−7 M) or lysosomal enzymes
(tritosomes), with rates being three times higher for linear polymers
than for polymers with the HMW star structure [33]. Almost no release
was observed in serum. For this biodistribution study, we used
conjugates containing GFLG spacers rather than the recently developed
conjugates with DOX bound via a pH-sensitive hydrazone bond [33]
even though these conjugates are highly effective in vivo; because of
their low stability in circulating blood, they are not as suitable for a bio-
distribution study.
y
x
O
O
O
HN
Gly
HN
HO
Phe
Leu
+
Gly
NH
OH
PAMAM
dendrimer
O
OH
O
O
O
O
3.2. Tumour and liver accumulation, blood clearance and excretion in
urine studies
HO
HO
O
OH
Tissue accumulation, blood clearance, and excretion in urine of
doxorubicin injected in the form of water-soluble polymer-DOX con-
jugates differing in molecular weights, polydispersities and molecular
architecture were studied in B6 mice bearing EL4 T-cell lymphoma.
Our aim was to select the polymer-drug carrier with the most suitable
structure for designing nanomedicines intended for the treatment of
solid tumours.
HoBT, DCC
DMF
3.2.1. Tumour and liver accumulation
Biodistribution study of HMW star conjugates with Mw above
200,000 g.mol−1 showed efficient tumour accumulation [39,45].
One of the aims of this study consisted in comparison of behaviour
GFLG Dox
of linear and star conjugates with lower Mw (≤100,000 g.mol−1
)
and verification of ability of the star conjugates to be accumulated
in tumours with similar efficiency as their HMW analogues. Tumour
accumulation of free doxorubicin (DOX.HCl) and its polymer conju-
gates 4–6 and 8–9 are compared in Fig. 3. All polymer conjugates
show significantly higher tumour accumulation in comparison to
DOX.HCl; however, HMW conjugates 5, 6, 8 and 9, with MWs higher
than the kidney threshold, displayed considerable tumour accumula-
tion in comparison to polymer conjugate 4, which has a MW below
this limit. Interestingly, no significant effect of conjugate polydisper-
sity (compare conjugates 5 and 6) on tumour accumulation was ob-
served. Therefore, prolonged tumour retention of the polymer
conjugates can only be attributed to a defective or nonexistent lym-
phatic system [6,46]. The highest amount of accumulated DOX was
achieved after administration of star polymer conjugate 9, while the
GFLG Dox
Dox GLFG
GFLG Dox
GFLG Dox
Dox GLFG
GFLG Dox
Fig. 2. Schematic description of the synthesis of star polymer-DOX conjugates 8 and 9.
weights of the resulting star polymers reflected that, on average, four
semitelechelic polymers were attached to the dendrimer core in star
polymer conjugate 8, and six polymer chains were attached to poly-
mer conjugate 9.
The hydrodynamic radii (Rh) of the linear and star polymer-DOX
conjugate coils in aqueous solution increased from ~5 nm to almost
14 nm depending on molecular weight. As expected, the size of
HMW linear conjugate 12 was slightly higher than it would corre-
spond with the size of branched star conjugates of the same molecu-
lar weights.
Fig. 3. Tumour accumulations in three C57BL/6 mice bearing EL4 lymphoma after i.v. in-
jection of DOX.HCl (5 mg/kg) or conjugates 4–6 or 8–9 (15 mg DOX eq./kg, an equitoxic
dose): (○ —) DOX·HCl, (□ —) conjugate 4, (Δ —) conjugate 5, (●—) conjugate 6, (▲ —)
conjugate 8, and (■ —) conjugate 9.
As previously described, DOX release from HPMA-based polymer con-
jugates containing DOX attached via oligopeptide spacers is controlled by

T. Etrych et al. / Journal of Controlled Release 164 (2012) 346–354
351
100
accumulation of DOX from linear conjugates 5 and 6 with comparable
90
80
70
60
50
40
30
20
10
0
molecular weights was lower. The time needed to reach maximum
tumour DOX accumulation exceeded 10 h and did not depend on
the architecture of polymer conjugates. Star polymer conjugates
8 and 9 seemed to accumulate in the tumour more slowly than linear
conjugates. It also seems that the molecular weight of star conjugates,
once exceeding renal threshold, do not significantly influence tumour
accumulation (compare with Ref. [45]). In all cases, the drug remains
accumulated in the tumour for a long period of time.
Liver accumulations of DOX.HCl and polymer conjugates 4–6 and
8–9 (Fig. 4) showed that the amount of polymer conjugates 4–6 and
8–9 in the liver exhibits a similar dependency on molecular weight
as previously shown for tumour accumulation. In contrast to tumour
accumulation, however, the concentration of polymer conjugates 4–6
and 8–9 in the liver increased during the first 24 h and decreased dur-
ing the following 48 h due to an efficient lymphatic and, perhaps,
hepatobiliary elimination system, which confirms previously pub-
lished conclusions [6,46].
0
20
40
time [h]
60
Fig. 5. Blood clearance in five C57BL/6 mice bearing EL4 lymphoma after i.v. injection of
DOX.HCl (5 mg/kg) or conjugates 4–6 or 8–9 (15 mg DOX eq./kg, an equitoxic dose):
(○ —) DOX·HCl, (□ —) conjugate 4, (Δ —) conjugate 5, (●—) conjugate 6, (▲ —) conjugate
8, and (■ —) conjugate 9.
3.2.2. Blood clearance
circulation and urine is negligible and practically all DOX is in its poly-
meric form (results not shown).
Blood clearance of linear conjugates 4–6 and star conjugates
8 and 9 was compared to that of free DOX.HCl (Fig. 5). The
low-molecular-weight DOX.HCl cleared from the bloodstream
very rapidly, being significantly faster than its polymer conjugates.
As expected, polymer architecture, hydrodynamic radius (Rh) and
weight average molecular weight (Mw) of each conjugate signifi-
cantly influenced the rate of elimination from blood circulation.
Blood clearance of linear polymer conjugates 5 and 6 and star poly-
mer conjugates 8 and 9, with molecular weights above the kidney
elimination threshold, were much slower than that of linear conju-
gate 4, which could be more easily removed from circulation by
glomerular filtration [21,22]. Interestingly, these results agree
with the cumulative amount of conjugates 4–6 and 8–9 found in
urine fractions collected within a 72 h period after the administra-
tion of the conjugates (Fig. 6).
Characteristics of the polymers (conjugates 4–6, 8, and 9) eliminat-
ed from the body through kidney clearance collected in urine within
72 h or 96 h after i.v. administration are summarised in Tables 2 and
3. The results show that each polymer is not eliminated as a whole
but rather as polymer fractions with very narrow molecular weight dis-
tribution; the polymers with the lowest molecular weights were excret-
ed first, followed by the higher molecular weight fractions (Table 2).
This phenomenon was observed for both the linear and star polymer
conjugates. After 72 h, the molecular weight of the polymer eliminated
by renal filtration reached almost 70,000 g.mol−1 for linear polymer
conjugates 4–6, which can be considered the threshold limit for elimi-
nation of linear HPMA conjugates through the kidney.
In contrast, for star conjugates 8 and 9, the threshold limit was ap-
proximately 50,000 g.mol−1 (see Table 3), which is in agreement
with the literature [47]. The differences observed in excretion limits
can be attributed to a different mechanism of polymer passage
through the kidney glomerulus. Filtration of the highly branched
star polymers are primarily controlled by the diameter of the relative-
ly rigid polymer coil in solution, while the limit for linear molecules
may be higher; transport of even larger polymer chains can be im-
proved by possible worm-like movement of such molecules through
the pores of glomerulus. Of course, such transport is slower than sim-
ple filtration and comes into effect in later stages of polymer elimina-
tion (36–96 h).
3.2.3. Excretion of the polymers in urine
The amount of polymer conjugate eliminated through the kidney in
urine fractions was determined by two methods. The first method cal-
culated the amount of polymer in urine fractions from the content of
DOX without characterising the excreted polymer. The second method
(HPLC) enabled the calculation of the amount of polymer-DOX in the
urine and molecular characterisation of the conjugate. The calculated
amounts of polymer-DOX conjugate in urine fractions, determined by
both independent methods, were in good agreement (Table 2). It is
understandable because DOX release from the conjugates in blood
It is also clear that the amount of excreted polymer depends on
the polydispersity of the original conjugate; more disperse conjugates
contain a higher portion of shorter polymer chains that can be filtered
100
90
80
70
60
50
40
30
20
10
0
conjugate 4 conjugate 5 conjugate 6 conjugate 8 conjugate 9
Fig. 4. Liver accumulations in three C57BL/6 mice bearing EL4 lymphoma after i.v. in-
jection of DOX.HCl (5 mg/kg) or conjugates 4–6 or 8–9 (15 mg DOX eq./kg, an
equitoxic dose): (○ —) DOX·HCl, (□ —) conjugate 4, (Δ —) conjugate 5, and (●—) con-
jugate 6, (▲ —) conjugate 8, and (■ —) conjugate 9.
Fig. 6. Cumulative urine eliminations of linear conjugates 4–6 and star conjugates 8–9
from C57BL/6 mice (5 in group) bearing EL4 lymphoma (i.v. injection; 72 h). 15 mg
DOX eq./kg was injected (i.v.).

352
T. Etrych et al. / Journal of Controlled Release 164 (2012) 346–354
Table 2
Characteristics of linear polymer conjugates isolated from urine.
Conjugate 4
Conjugate 5
Conjugate 6
Time (h)
Mw (g.mol−1
)
Mw/Mn
% (a/b)
Mw (g.mol−1
)
Mw/Mn
% (a/b)
Mw (g.mol−1
)
Mw/Mn
%
1
3
6
9
12
24
30
36
48
72
19,100
22,000
28,000
28,800
33,000
39,100
41,500
48,000
53,000
68,000
1.05
1.06
1.05
1.07
1.20
1.10
1.12
1.10
1.32
1.20
38.7/35.7
24.7/26.1
3.1/4.1
2.6/3.6
3.2/4.1
3.9/3.2
1.1/0.4
0.8/0.3
0.8/0.3
1.4/0.4
29,800
33,000
39,000
41,500
50,500
51,000
62,300
66,000
68,800
69,000
1.04
1.06
1.05
1.08
1.11
1.17
1.14
1.16
1.19
1.22
4.3/4.8
2.8/3.6
1.2/1.7
1.0/1.3
1.0/1.0
1.5/0.9
0.9/0.3
0.5/0.2
0.5/0.2
0.1/0.4
23,950
26,700
31,000
32,300
35,100
36,000
40,200
49,200
53,200
69,100
1.07
1.07
1.05
1.06
1.05
1.07
1.14
1.25
1.26
1.32
8.8/8.3
6.8/6.4
1.5/1.2
0.5/0.6
1.4/1.4
1.4/1.2
0.5/0.3
0.1/0.3
0.8/0.4
0.1/0.3
a
Percent of polymer in the urine sample determined by the DOX content.
Percent of polymer in the urine sample determined by GPC via dn/dc.
b
in an initial phase of polymer excretion (compare conjugates 5 and 6,
Table 3).
polymer conjugate (Fig. 7) with DOX bound via a GFLG spacer (conju-
gate 10) or with the similar conjugate bearing DOX bound via
pH-sensitive hydrazone bond (conjugate 11) resulted in complete tu-
mour regression of all animals, as 100% of animals were considered
long-term surviving (LTS). A decrease in the dose to 10 mg DOX eq./
kg resulted in lower efficacy of treatment, resulting in 75% LTS for con-
jugate 11 (hydrazone bond) and 63% for conjugate 10 (amide bond). A
further decrease in the dose to 5 mg DOX eq./kg produced only 37% LTS
in case of conjugate 11 and no LTS for conjugate 10. Conjugates con-
taining the GFLG spacer and amide bond are somewhat less effective
than their corresponding conjugates containing the pH-sensitive
hydrazone bond, but this difference is relatively small. Animals treated
with 10 mg DOX eq./kg of a HMW linear conjugate 12 (Fig. 8) exhibited
similar survival (75% LTS) as respective star conjugate, while treatment
with conjugate 4 (Mw 36 000 g.mol−1) was much less effective, thus in-
dicating the importance of the EPR effect on final anti‐tumour activity.
The survival data shown above are in good agreement with tumour
growth/regression data shown as supplementary in Figs. 9 and 10
(see supplement). The results shown in Figs. 7 and 8 indicate that
anti‐tumour activity of the conjugates depends more on the size of
their coil in solution (Rh of HMW conjugates ~13–14 nm, Rh of conju-
gate 4–4.5) than on molecular weight and detailed polymer structure.
The results of our evaluation of anti-cancer activity of HPMA co-
polymer conjugates show that the HMW star conjugates with the
lowest blood clearance rate and the highest tumour accumulation ex-
hibit the highest anti‐tumour activity in vivo, with complete tumour
regression of animals at 15 mg DOX eq./kg, and this activity is
The cumulative amounts of polymer conjugates 4–6, 8, and 9 eliminat-
ed by renal filtration are shown in Fig. 6. After 72 h, 80% of polymer con-
jugate 4, with the lowest molecular weight, was eliminated by renal
filtration; conjugates 5 and 6, with higher molecular weights, exhibited
lower cumulative amounts eliminated by the kidneys, with the more
polydisperse conjugate 6 cleared from the blood faster (Fig. 5) and excret-
ed in the urine to a larger extent (25%) than monodisperse conjugate 5
(15%). The cumulative amounts of HMW star polymer conjugates 8 and
9 eliminated by renal filtration were lower than those amounts for conju-
gates 4–6 of similar molecular weights, demonstrating the influences of a
more rigid molecular architecture. Therefore, it is clear that complete
elimination of the HMW linear and star polymer conjugates intended
for in vivo treatment can only be achieved by designing polymer carrier
systems containing biodegradable linkages enabling polymer carrier frag-
mentation to sequences with molecular weights sufficiently below kidney
threshold [33,45].
3.3. In vivo anti-cancer activity
Anti-tumour activity of linear and star HPMA copolymer-DOX
conjugates was tested in C57BL/6 (B/6) mice with s.c. implanted mu-
rine syngeneic tumour model EL4 T-cell lymphoma (1×105). The ef-
fect of HMW conjugates with Rh ~13 nm and conjugates with lower
Mw (conjugate 4) were evaluated. eq./kg.
The mice (8 per group) were treated i.v. on day 8 after cell transplan-
tation with a single dose of the conjugates, either 15, 10 or 5 mg DOX
No weight loss or other signs of acute toxicity (data not shown) were
observed after conjugate injection. In most cases, inhibition or even re-
gression of tumour growth was observed. Overall survival of mice after
treatment with star (Fig. 7) or linear (Fig. 8) HPMA copolymer-DOX
conjugates depended on the dose and molecular weight of the conju-
gate. Treatment of mice with 15 mg DOX eq./kg of the HMW star
100
80
60
40
20
0
Table 3
Characteristics of star polymer conjugates isolated from urine.
Time (h)
Conjugate 8
% (a)
Conjugate 9
% (a)
Mw (g.mol−1
)
Mw/Mn
Mw (g.mol−1
)
Mw/Mn
0
10
20
30
40
days
50
60
70
1
3
6
12
24
48
72
96
22,650
25,600
33,650
38,100
38,000
44,000
49,000
51,200
1.06
1.08
1.03
1.02
1.03
1.12
1.05
1.04
3.9
3.5
2.3
1.8
1.8
1.6
1.6
2.0
43,000
44,800
47,000
47,000
48,000
50,300
52,000
53,300
1.14
1.07
1.04
1.08
1.05
1.06
1.08
1.15
1.8
0.8
0.6
0.8
0.8
0.8
0.6
0.3
Fig. 7. Survival of EL4 T-cell lymphoma-bearing B/6 mice treated with star
polymer-DOX conjugates. B/6 mice were transplanted s.c. with 1×105 EL4 T-cells
and treated with (○- - -) DOX.HCl, two doses, 5 mg DOX/kg each; (♦ —) conjugate
11, 1×15 mg DOX eq./kg; (■— ) conjugate 11, 1×10 mg DOX eq./kg; (▲- - -) conju-
gate 11, 1×5 mg DOX eq./kg; (Δ - - -) conjugate 10, 1×15 mg DOX eq./kg; (○—) con-
jugate 10, 1×10 mg DOX eq./kg; using i.v. drug administration on day 8. Control mice
(—) were left untreated.
a
Percent of polymer in the urine sample determined by the DOX content.

T. Etrych et al. / Journal of Controlled Release 164 (2012) 346–354
353
Appendix A. Supplementary data
100
80
60
40
20
0
Supplementary data to this article can be found online at http://
dx.doi.org/10.1016/j.jconrel.2012.06.029.
References
[1] G. Pasut, M. Sergi, F.M. Veronese, Anti-cancer PEG-enzymes: 30 years old, but still
a current approach, Adv. Drug Deliv. Rev. 60 (2008) 69–78.
[2] R. Duncan, Polymer conjugates as anticancer nanomedicines, Nat. Rev. Cancer 6
(2006) 688–701.
0
10
20
30
40
50
60
70
[3] J. Kopeček, Biomaterials and drug delivery: past, present, and future, Mol. Pharm.
days
7 (2010) 922–925.
[4] B. Říhová, L. Kovář, M. Kovář, O. Hovorka, Cytotoxicity and immunostimulation:
double attack on cancer cells with polymeric therapeutics, Trends Biotechnol.
27 (2009) 11–17.
Fig. 8. Survival of EL4 T-cell lymphoma-bearing B/6 mice treated with linear
polymer-DOX conjugates. B/6 mice were transplanted s.c. with 1×105 EL4 T-cells
and treated with (♦- - -) conjugate 12, 1×10 mg DOX eq./kg or (▲- - -) conjugate 4,
1×10 mg DOX eq./kg using i.v. drug administration on day 8. Control mice (—) were
left untreated.
[5] B. Říhová, M. Kovář, Immunogenicity and immunomodulatory properties of
HPMA-based polymers, Adv. Drug Deliv. Rev. 62 (2010) 184–191.
[6] Y. Matsumura, H. Maeda, A new concept for macromolecular therapeutics in
cancer-chemotherapy — mechanism of tumoritropic accumulation of proteins
and the antitumor agent Smancs, Cancer Res. 46 (1986) 6387–6392.
[7] H. Maeda, K. Greish, J. Fang, The EPR effect and polymeric drugs: a paradigm shift for
cancer chemotherapy in the 21st century, In: in: R. Satchi-Fainaro, R. Duncan (Eds.),
Advances in Polymer Science, Springer, Berlin/Heidelberg, 2006, pp. 103–121.
[8] H. Maeda, Tumor-selective delivery of macromolecular drugs via the EPR effect:
background and future prospects, Bioconjug. Chem. 21 (2010) 797–802.
[9] M.E. Fox, F.C. Szoka, J.M.J. Frechet, Soluble polymer carriers for the treatment of
cancer: the importance of molecular architecture, Acc. Chem. Res. 42 (2009)
1141–1151.
[10] H.G. Rennke, M.A. Venkatachalam, Glomerular-permeability of macromolecules —
effect of molecular-configuration on the fractional clearance of uncharged dextran
and neutral horseradish-peroxidase in the rat, J. Clin. Invest. 63 (1979) 713–717.
[11] M.A. Venkatachalam, H.G. Rennke, Structural and molecular-basis of
glomerular-filtration, Circ. Res. 43 (1978) 337–347.
[12] W.M. Deen, M.J. Lazzara, B.D. Myers, Structural determinants of glomerular per-
meability, Am. J. Physiol. Renal Physiol. 281 (2001) F579–F596.
[13] J. Tencer, I.M. Frick, B.W. Oquist, P. Alm, B. Rippe, Size-selectivity of the glomeru-
lar barrier to high molecular weight proteins: upper size limitations of shunt
pathways, Kidney Int. 53 (1998) 709–715.
practically independent on mode of drug attachment (e.g., enzymati-
cally or hydrolytically cleavable hydrazone spacers). This finding is
quite surprising because all experiments previously performed with
HPMA copolymer-doxorubicin conjugates with molecular weights
below the renal threshold have displayed much higher conjugate ac-
tivity when DOX was bound via hydrazone bond compared to conju-
gates containing a GFLG spacer; furthermore, such
a dramatic
anti-cancer effect in the case of GFLG spacer-containing conjugates
was unexpected (this was why conjugate 10 with Mw 248 kDa was
selected for in vivo evaluation to achieve higher activity than was
expected from conjugate 9 (94 kDa).
4. Conclusions
[14] D. Asgeirsson, D. Venturoli, E. Fries, B. Rippe, C. Rippe, Glomerular sieving of three
neutral polysaccharides, polyethylene oxide and bikunin in rat. Effects of molec-
ular size and conformation, Acta Physiol. 191 (2007) 237–246.
The present study describes the synthesis, physicochemical char-
acterisation and determination of biological properties, namely
blood clearance, kidney elimination, tumour and liver accumulation
and survival of mice bearing EL 4 T-cell lymphoma after i.v. injection
of HPMA copolymer-DOX conjugates. The conjugates differed in mo-
lecular weights, polydispersities and molecular architecture.
The biological properties of linear polymer-DOX conjugates with
different molecular weights and polydispersities were compared to
the properties of dendrimer core-based star polymer-DOX conjugates
with relatively rigid structures.
[15] D. Asgeirsson, J. Axelsson, C. Rippe, B. Rippe, Similarity of permeabilities for Ficoll,
pullulan, charge-modified albumin and native albumin across the rat peritoneal
membrane, Acta Physiol. 196 (2009) 427–433.
[16] N. Nasongkla, B. Chen, N. Macaraeg, M.E. Fox, J.M.J. Frechet, F.C. Szoka, Depen-
dence of pharmacokinetics and biodistribution on polymer architecture: effect
of cyclic versus linear polymers, J. Am. Chem. Soc. 131 (2009) 3842–3843.
[17] R. Rodewald, M.J. Karnovsky, Porous substructure of glomerular slit diaphragm in
rat and mouse, J. Cell Biol. 60 (1974) 423–433.
[18] A.C. Guyton, J.E. Hall, Textbook of Medicinal Physiology, In: Elsevier Saunders,
Philadelphia, 2006, pp. 316–317.
[19] S. Sadekar, A. Ray, M. Janat-Amsbury, C.M. Peterson, H. Ghandehari, Comparative bio-
distribution of PAMAM dendrimers and HPMA copolymers in ovarian-tumor-bearing
mice, Biomacromolecules 12 (2011) 88–96.
[20] Y. Noguchi, J. Wu, R. Duncan, J. Strohalm, K. Ulbrich, T. Akaike, H. Maeda, Early
phase tumor accumulation of macromolecules: a great difference in clearance
rate between tumor and normal tissues, Jpn. J. Cancer Res. 89 (1998) 307–314.
[21] M. Kissel, P. Peschke, V. Šubr, K. Ulbrich, J. Schuhmacher, J. Debus, E. Friedrich,
Synthetic macromolecular drug carriers: biodistribution of poly[N-(2-hydro-
xypropyl)methacrylamide] copolymers and their accumulation in solid rat tu-
mors, PDA J. Pharm. Sci. Technol. 55 (2001) 191–201.
[22] T. Lammers, R. Kuhnlein, M. Kissel, V. Šubr, T. Etrych, R. Pola, M. Pechar, K. Ulbrich,
G. Storm, P. Huber, P. Peschke, Effect of physicochemical modification on the bio-
distribution and tumor accumulation of HPMA copolymers, J. Control. Release
110 (2005) 103–118.
[23] V. Šubr, J. Strohalm, K. Ulbrich, R. Duncan, I.C. Hume, Polymers containing enzy-
matically degradable bonds, XII. Effect of spacer structure on the rate of release
of daunomycin and adriamycin from poly-[2-(hydroxypropyl)methacrylamide]
drug carriers in vitro and antitumour activity measured in vivo, J. Control. Release
18 (1992) 123–132.
[24] T. Etrych, M. Jelínková, B. Říhová, K. Ulbrich, New HPMA copolymers containing
doxorubicin bound via pH-sensitive linkage: synthesis and preliminary in vitro
and in vivo biological properties, J. Control. Release 73 (2001) 89–102.
[25] R. Duncan, N-(2-Hydroxypropyl)methacrylamide copolymer conjugates, In: in:
G.S. Kwon (Ed.), Drugs and the Pharmaceutical Sciences, Polymeric Drug Delivery
Systems, vol. 148, Taylor & Francis, Boca Raton, 2005, pp. 1–92.
The molecular weights of the linear polymer-DOX conjugates ex-
creted in urine of mice by renal filtration were up to 70,000 g.mol−1
,
while the highest molecular weight of the star conjugates excreted in
urine was ~50,000 g.mol−1. The amount of polymer-DOX conjugate
eliminated by renal filtration decreased with increasing molecular
weight, and linear conjugates with comparable molecular weights to
the star conjugates were eliminated more quickly. Polymers were not
excreted in urine as the whole polymer fraction with molecular weights
below 50,000 g.mol−1 but were rather excreted as narrow polymer
fractions with molecular weights gradually increasing with time follow-
ing injection (excreted urine volume). Finally, polymer-DOX conjugates
with higher molecular weights exhibited longer blood circulation times
and higher tumour accumulations, which resulted in significantly im-
proved anti‐tumour activities of the HMW conjugates in vivo, indepen-
dent of drug attachment form.
Acknowledgements
[26] K. Ulbrich, V. Šubr, Synthetic polymer-drug conjugates for human therapy, In: in:
M.M. Amiji, V.P. Torchilin (Eds.), Handbook of Materials for Nanomedicine, vol.1,
Pan Stanford Publishing, 2010, pp. 5–79.
[27] R. Duncan, M.J. Vicent, Do HPMA copolymer conjugates have a future as clinically
useful nanomedicines? A critical overview of current status and future opportuni-
ties, Adv. Drug Deliv. Rev. 62 (2010) 272–282.
This work was supported by the grant agency of the Academy of Sci-
ences of the Czech Republic (grant no. IAAX00500803) and the grant
agency of the Czech Republic (grant no. P301/11/0325).

354
T. Etrych et al. / Journal of Controlled Release 164 (2012) 346–354
[28] L.W. Seymour, D.R. Ferry, D.J. Kerr, D. Rea, M. Whitlock, R. Poyner, Ch. Boivin,
S. Hesslewood, Ch. Twelves, R. Blackie, A. Schatzlein, D. Jodrell, D. Bissett, H.
Calvert, M. Lind, A. Robbins, S. Burtles, R. Duncan, J. Cassidy, Phase II studies
of polymer-doxorubicin (PK1, FCE28068) in the treatment of breast, lung
and colorectal cancer, Int. J. Oncol. 34 (2009) 1629–1636.
marrow in vivo and mouse splenocytes and human peripheral blood lympho-
cytes in vitro, Biomaterials 10 (1989) 335–342.
[39] T. Etrych, J. Strohalm, P. Chytil, B. Říhová, K. Ulbrich, Novel star HPMA-based poly-
mer conjugates for passive targeting to solid tumors, J. Drug Target. 19 (2011)
874–889.
[29] B. Říhová, K. Kubáčková, Clinical implications of N-(2-hydroxypropyl)methacrylamide
[40] D. Fraier, E. Frigerio, E. Pianezzola, M.S. Benedetti, J. Cassidy, P. Vasey, A sensi-
tive procedure for the quantitation of free and N-(2-hydroxypropyl)met-
hacrylamide polymer-bound doxorubicin (Pk1) and some of its metabolites,
13-dihydrodoxorubicin, 13-dihydrodoxorubicinone and doxorubicinone, in
human plasma and urine by reversed-phase Hplc with fluorometric detection,
J. Pharm. Biomed. Anal. 13 (1995) 625–633.
[41] D. Wang, P. Kopečková, T. Minko, V. Nanayakkara, J. Kopeček, Synthesis of starlike
N-(2-hydroxypropyl)methacrylamide copolymers: potential drug carriers, Bio-
macromolecules 1 (2000) 313–319.
[42] T. Etrych, L. Kovář, V. Šubr, A. Braunová, M. Pechar, P. Chytil, B. Říhová, K. Ulbrich,
High-molecular-weight polymers containing biodegradable disulfide bonds: syn-
thesis and in vitro verification of intracellular degradation, J. Bioact. Compat.
Polym. 25 (2010) 5–26.
[43] L.W. Seymour, Y. Miyamoto, H. Maeda, M. Brereton, J. Strohalm, K. Ulbrich, R. Duncan,
Influence of molecular weight on passive tumour accumulation of a soluble macro-
molecular drug carrier, Eur. J. Cancer 31A (1995) 766–770.
[44] P. Rejmanová, J. Kopeček, J. Pohl, M. Baudyš, V. Kostka, Polymers containing en-
zymatically degradable bonds. 8. Degradation of oligopeptide sequences in
N-(2- hydroxypropyl)methacrylamide copolymers by bovine spleen cathep-
sin B, Makromol. Chem. 184 (1983) 2009–2020.
[45] T. Etrych, L. Kovář, J. Strohalm, P. Chytil, B. Říhová, K. Ulbrich, Biodegradable star
HPMA polymer-drug conjugates: biodegradability, distribution and anti-tumor
efficacy, J. Control. Release 154 (2011) 241–248.
[46] H. Maeda, J. Wu, T. Sawa, Y. Matsumura, K. Hori, Tumor vascular permeability and
the EPR effect in macromolecular therapeutics: a review, J. Control. Release 65
(2000) 271–284.
[47] L.W. Seymour, R. Duncan, J. Strohalm, J. Kopeček, Effect of molecular weight (Mw)
of N-(2-hydroxypropyl)methacrylamide copolymers on body distribution and
rate of excretion after subcutaneous intraperitoneal and intravenous administra-
tion to rats, J. Biomed. Mater. Res. 21 (1987) 1341–1358.
copolymers, Curr. Pharm. Biotechnol. 4 (2003) 311–322.
[30] B. Říhová, Clinical experience with anthracycline antibiotics-HPMA copolymer-human
immunoglobulin conjugates, Adv. Drug Deliv. Rev. 61 (2009) 1149–1158.
[31] T. Etrych, P. Chytil, T. Mrkvan, M. Šírová, B. Říhová, K. Ulbrich, Conjugates of doxo-
rubicin with graft HPMA copolymers for passive tumor targeting, J. Control. Re-
lease 132 (2008) 184–192.
[32] P. Chytil, T. Etrych, Č. Koňák, M. Šírová, T. Mrkvan, J. Bouček, B. Říhová, K.
Ulbrich, New HPMA copolymer-based drug carriers with covalently bound hy-
drophobic substituents for solid tumour targeting, J. Control. Release 127
(2008) 121–130.
[33] T. Etrych, J. Strohalm, P. Chytil, P. Černoch, L. Starovoytova, M. Pechar, K. Ulbrich,
Biodegradable star HPMA polymer conjugates of doxorubicin for passive tumor
targeting, Eur. J. Pharm. Sci. 42 (2011) 527–539.
[34] P. Rejmanová, J. Kopeček, R. Duncan, J.B. Lloyd, Stability in rat plasma and serum
of lysosomally degradable oligopeptide sequences in N-(2-hydroxypropyl)met-
hacrylamide copolymers, Biomaterials 6 (1985) 45–48.
[35] R. Duncan, P. Kopečková-Rejmanová, J. Strohalm, I.C. Hume, H.C. Cable, J. Pohl, J.B.
Lloyd, J. Kopeček, Anticancer agents coupled to N-(2-hydroxypropyl)methacrylamide
copolymers. 1. Evaluation of daunomycin and puromycin conjugates in vitro, Br. J. Can-
cer 55 (1987) 165–174.
[36] K. Ulbrich, V. Šubr, J. Strohalm, D. Plocová, M. Jelínková, B. Říhová, Polymeric
drugs based on conjugates of synthetic and natural macromolecules I. Synthesis
and physico-chemical characterisation, J. Control. Release 64 (2000) 63–79.
[37] V. Šubr, K. Ulbrich, Synthesis and properties of new N-(2-hydroxypropyl)met-
hacrylamide copolymers containing thiazolidine-2-thione reactive groups,
React. Funct. Polym. 66 (2006) 1525–1538.
[38] B. Říhová, K. Ulbrich, J. Strohalm, V. Vetvička, M. Bilej, J. Kopeček, R. Duncan, Bio-
compatibility of N-2-(hydroxypropyl)methacrylamide copolymers containing
adriamycin. Immunogenicity, and effect of haematopoietic stem cells in bone