Backbone degradable multiblock N-(2-hydroxypropyl)methacrylamide copolymer conjugates via reversible addition-fragmentation chain transfer polymerization and thiol-ene coupling reaction

Article abstract of DOI:10.1021/bm101254e

Telechelic water-soluble HPMA copolymers and HPMA copolymer-doxorubicin (DOX) conjugates have been synthesized by RAFT polymerization mediated by a new bifunctional chain transfer agent (CTA) that contains an enzymatically degradable oligopeptide sequence. Postpolymerization aminolysis followed by chain extension with a bis-maleimide resulted in linear high molecular weight multiblock HPMA copolymer conjugates. These polymers are enzymatically degradable; in addition to releasing the drug (DOX), the degradation of the polymer backbone resulted in products with molecular weights similar to the starting material and below the renal threshold. The new multiblock HPMA copolymers hold potential as new carriers of anticancer drugs.

Full text of DOI:10.1021/bm101254e

Biomacromolecules 2011, 12, 247–252
247
Backbone Degradable Multiblock
N-(2-Hydroxypropyl)methacrylamide Copolymer Conjugates via
Reversible Addition-Fragmentation Chain Transfer
Polymerization and Thiol-ene Coupling Reaction
Huaizhong Pan, Jiyuan Yang, Pavla Kopecˇkova´, and Jindrˇich Kopecˇek*
Departments of Pharmaceutics and Pharmaceutical Chemistry, and of Bioengineering, University of Utah,
Salt Lake City, Utah 84112, United States
Received October 20, 2010; Revised Manuscript Received November 25, 2010
Telechelic water-soluble HPMA copolymers and HPMA copolymer-doxorubicin (DOX) conjugates have been
synthesized by RAFT polymerization mediated by a new bifunctional chain transfer agent (CTA) that contains an
enzymatically degradable oligopeptide sequence. Postpolymerization aminolysis followed by chain extension with
a bis-maleimide resulted in linear high molecular weight multiblock HPMA copolymer conjugates. These polymers
are enzymatically degradable; in addition to releasing the drug (DOX), the degradation of the polymer backbone
resulted in products with molecular weights similar to the starting material and below the renal threshold. The
new multiblock HPMA copolymers hold potential as new carriers of anticancer drugs.
concomitant increase in therapeutic efficacy. But this method
was limited by poor structural control and reproducibility, and
steric hindrance for enzyme hydrolysis. To overcome these
shortcomings, biodegradable HPMA copolymers with well
controlled structures should be designed.
RAFT polymerization has proven to be an extremely well
controlled “living” free radical polymerization method for the
preparation of polymers with designed molecular weights and
narrow molecular weight distribution.9,10 It permits the synthesis
of telechelic polymers11 as well as providing easy modifica-
tion of terminal groups. For example, postpolymerization
aminolysis of thiobenzoylthio end groups results in R,ω-dithiol
telechelic polymers.12,13 Biodegradable, long-circulating polymer
carriers can be prepared by linking 10-40 kDa linear, telechelic
polymer segments (synthesized by RAFT polymerization, with
molecular weights below the renal threshold) to enzymatically
degradable oligopeptide sequences.
Here we report the design and synthesis of novel lysosomally
degradable water-soluble multisegment HPMA copolymers via
the combination of reversible addition-fragmentation chain
transfer (RAFT) polymerization and high efficient thiol-ene
reaction. The structure and molecular weight of each segment
were controlled by RAFT polymerization, and the incorporation
of the enzymatically degradable sequences into the linear
polymer backbone provides for an easy formation of the
enzyme-substrate complex, resulting in fast degradation.
Introduction
Copolymers of N-(2-hydroxypropyl)methacrylamide (HPMA)
have been some of the most frequently studied water-soluble
carriers of anticancer drugs.1,2 Their biological evaluation both
in animal models and clinical trials revealed their biocompat-
ibility.2 The molecular weight of the drug carriers is crucial for
effective functioning. The molecular weight threshold limiting
glomerular filtration of poly[N-(2-hydroxypropyl)methacryl-
amide] (polyHPMA) is approximately 45 kDa for intravenous
administration; consequently, the molecular weight of polymeric
carriers should be less than 30-40 kDa.3 For example, poly-
HPMA-GFLG-doxorubicin, PK1, had an average molecular
weight of 28 kDa and a hydrodynamic diameter about 5 nm,
with an initial half-life of 2.7 h.4 This resulted in short circulation
times and limited accumulation in the tumor tissue.5,6 The
molecular weight of currently used HPMA copolymer-drug
conjugates is suboptimal.5 Increasing the molecular weight of
HPMA copolymers resulted in prolonged circulation times and
increased the tumor-to-organ ratios due to the EPR (enhanced
permeability and retention) effect.7 However, high molecular
weight nonbiodegradable HPMA copolymer conjugates may
deposit inside the body and cause unexpected side effects. The
lack of polymer biodegradability will likely limit parenteral use
of HPMA copolymer conjugates as treatments for life-threaten-
ing diseases.6 Biodegradable polyesters such as PLGA (poly-
(lactic-co-glycolic acid)) are used in drug delivery systems, but
they are limited by their hydrophobicity and/or nonspecific
degradation. HPMA copolymers that can be biodegraded in
cells, but stable in blood circulation are needed.
Experimental Section
Materials and Methods. N-R-Fmoc protected amino acids, 2-(1H-
benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate
(HBTU) and 2-Cl-trityl chloride resin (100-200 mesh, 1.27 mmol/g)
were from EMD Biosciences (San Diego, CA). Papain (EC 3.4.22.2,
from papaya latex) and cathepsin B (EC 3.4.22.1, from bovine spleen)
were from Sigma-Aldrich (St. Louis, MO). Bis-MAL-dPEG3 (bis-[1,13-
(3-maleimidopropionyl)amido]-4,7,10-trioxatridecane, CAS No. 756525-
89-0) was purchased from Quanta Biodesign (Powell, OH). 1-Hy-
droxybenzotriazole (HOBt) was purchased from AnaSpec (Fremont,
CA). N,N′-Diisopropylcarbodiimide (DIC), 2,2,2-trifluoroethanol (TFE),
Shiah et al.8 synthesized biodegradable HPMA copolymer-
DOX conjugates by slightly cross-linking of copolymer chains
via enzymatic cleavable -GFLG- tetrapeptide and tested the
conjugates in mice bearing human ovarian xenografts. The
results clearly indicated that the higher the molecular weight
of the carrier, the higher the accumulation in solid tumor with
* To whom correspondence should be addressed. E-mail:
jindrich.kopecek@utah.edu.
10.1021/bm101254e
 2011 American Chemical Society
Published on Web 12/15/2010

248 Biomacromolecules, Vol. 12, No. 1, 2011
Pan et al.
and all other reagents and solvents were from Sigma-Aldrich (St. Louis,
MO). Doxorubicin (DOX) was a kind gift from Meiji Seika Kaisha
Ltd. Tokyo, Japan. HPMA,14 4-cyanopentanoic acid dithiobenzoate15
were synthesized according to literature. N-Methacryloylglycylphenyl-
alanylleucylglycyl-doxorubicin (MA-GFLG-DOX) was prepared by the
reaction N-methacryloylglycylphenylalanylleucylglycine 4-nitrophenyl
ester (MA-GFLG-ONp) with doxorubicin hydrochloride in DMF in
the presence of diisopropylethylamine according to the described
procedure.16
acetone three times, then washed with acetone, and dried under reduced
pressure at room temperature. Yield 145 mg (72.5%) of pink R,ω-
di(phenylcarbonothioylthio)-polyHPMA (Mw 30.3 kDa, PDI 1.08).
Kinetic Studies. Stock solution was prepared by dissolving HPMA
(720 mg, 5 mmol), peptide2CTA (17.7 mg, 12.5 µmol), and AIBN
(0.4 mg, 2.4 µmol) in methanol (4 mL) in a vial equipped with a rubber
septum and then bubbled with N2 for 30 min. Aliquots were transferred
via syringe to six different ampules; each one was attached to Schlenk-
line and had been purged with N2. The ampules were sealed under N2
atmosphere and then placed in an oil bath preheated to 60 °C. At
different time points, the ampule was taken out and the polymerization
quenched in liquid nitrogen. The sample solution was immediately
analyzed by HPLC. The monomer conversion was calculated by
comparison of the remaining monomer concentration with initial
concentration. The molecular weight of the sample was measured by
FPLC using Superose 12 HR10/30 column with PBS (pH 7.3) as the
mobile phase after precipitation of the polymer solution into cold
anhydrous ether.
UV-vis spectra were measured on a Varian Cary 400 Bio
UV-visible spectrophotometer. Mass spectra were measured on an
FTMS mass spectrometer (LTQ-FT, ThermoElectron, Waltham, MA).
1H NMR spectra were recorded on a Mercury400 spectrometer using
DMSO-d6 as the solvent. Polymerization conversion was determined
by the measurement of remaining HPMA monomer concentration at
different time points using RP-HPLC (Agilent Technologies 1100 series,
Zorbax C8 column 4.6 × 150 mm) with gradient elution from 2 to
90% of Buffer B within 30 min at flow rate of 1.0 mL/min (Buffer A:
deionized water (DI H2O) with 0.1% TFA; Buffer B: acetonitrile with
0.1% TFA). The molecular weight and polydispersity index (PDI) of
Measurement of the Molecular Weight of the PolyHPMA by
Extinction Coefficient of Peptide2CTA. A series of peptide2CTA solutions
in methanol ranging from 6.33 × 10-6 M to 6.33 × 10-5 M were prepared.
The absorbance of each sample at 302 nm (λmax PhCdSS) was measured
using a 1.0 cm path length quartz cuvette. The molar absorption coefficient
(ε, in methanol) of 2.69 × 104 M-1 cm-1 was obtained from the slope of
¨
polymers were measured on an AKTA FPLC (fast protein liquid
chromatography) system (GE Healthcare, formerly Amersham) equipped
with miniDAWN TREOS and OptilabEX detectors (Wyatt Technology,
Santa Barbara, CA) using a Superose 6 or 12 HR10/30 column with
PBS (pH 7.3) as the mobile phase. Narrow polydispersity polyHPMA
fractions prepared by size exclusion chromatography, whose molecular
weights were characterized by multiangle light scattering, were used
as molecular weight standards. The multiblock polymers were fraction-
ated on the same FPLC system using Superose 6 HR16/60 preparative
column. PBS was used as the mobile phase. The flow rate was 1 mL/
min the fraction was collected every 10 min. The salt in the fractions
was removed by dialysis. The narrow polydispersity polymer fractions
were obtained after freeze-drying.
Synthesis of Enzyme-Sensitive Bifunctional CTA, Nr,Nε-Bis(4-
cyano-4-(phenylcarbonothioylthio)pentanoylglycylphenylalanylleu-
cylglycyl)lysine (Peptide2CTA). The bifunctional chain transfer agent
containing an enzyme-sensitive peptide sequence was synthesized by
solid phase peptide synthesis (SPPS) methodology and manual Fmoc/
tBu strategy on 2-chlorotrityl chloride resin. HBTU was used as the
coupling agent and 20% piperidine in DMF as the deprotection agent
for Fmoc protected amino acids (Fmoc-AA-OH). Briefly, Fmoc
protected amino acids, Fmoc-Lys(Fmoc)-OH, Fmoc-Gly-OH, Fmoc-
Leu-OH, Fmoc-Phe-OH, and Fmoc-Gly-OH were coupled sequentially
to the beads (0.3 g, 0.11 mmol loading). After deprotection, the chain
transfer agent, 4-cyanopentanoic acid dithiobenzoate (3× excess), was
coupled to the terminal glycyl residues using DIC/HOBT (1/1, 3×
excess) in DMF. The peptide was isolated following cleavage from
resin by 30% TFE in DCM for 2 h. Yield 99 mg (62%). The purity of
peptide2CTA was measured by HPLC. MS (m/z): [M + H]+ 1417.56467
(calcd C70H88N12O12S4 + H, 1417.56003), [M + 2H]2+ 709.28516 (calcd
C70H88N12O12S4 + 2H, 709.28365). 1H NMR (DMSO-d6, δ, ppm): 12.62
(s, 1H, -COOH); 8.27-7.96 (m, 10H, CONH); 7.90 (d, 3H, Ph-H);
7.68 (t, 3H, Ph-H); 7.50 (t, 4H, Ph-H); 7.22 (m, 10H, Ph-H); 4.52 (m,
2H, Phe-CH); 4.25 (m, 2H, Leu-CH); 4.16 (m, 1H, Lys-CH); 3.73-3.55
(m, 8H, Gly-CH2); 3.01 (m, 4H, Phe-CHH, Lys-NH-CH2); 2.74 (m,
2H, Phe-CHH); 2.49-2.33 (m, 8H, CTA-CH2CH2); 1.88 (s, 6H, CTA-
CH3); 1.70-1(0.27 (m, 12H, Lys-C-CH2CH2CH2-C, Leu-
CH2CHMe2); 0.87-0.81 (dd, 12H, Leu-(CH3)2).
the plot Concentration (M) ∼ absorbance302 nm
.
Polymers obtained from the kinetics study were dissolved in
methanol. Similarly, the absorbance of each sample at 302 nm (λmax
PhCdSS) was measured using a 1.0 cm path length quartz cuvette.
The molecular weight of each polymer was calculated using the
extinction coefficient of peptide2CTA.
RAFT Copolymerization of HPMA with MA-GFLG-DOX. HPMA
copolymerization was performed similarly as the homopolymerization:
HPMA (140 mg, 977.7 µmol, 98 mol %), MA-GFLG-DOX (20 mg,
20.3 µmol, 2 mol %), peptide2CTA (5.7 mg, 4 µmol), and AIBN (0.4
mg, 2.4 µmol) were dissolved in methanol (1.0 mL). The solution was
bubbled with N2 for 30 min, sealed, and polymerized at 50 °C for 24 h.
After polymerization, the polymer was purified by dissolution-precipi-
tation in methanol-acetone three times, then washed with acetone, and
dried under reduced pressure at room temperature. Yield 100 mg
(62.5%) of R,ω-di(phenylcarbonothioylthio)-HPMA copolymer-DOX
conjugate (Mw 27.8 kDa, PDI 1.19).
End Group Modification by Aminolysis. R,ω-Di(phenylcarbono-
thioylthio)-polyHPMA (100 mg) was dissolved in 800 µL of methanol
(bubbled with N2 for 30 min); n-butylamine (100 µL) was added under
N2 and the mixture was stirred for 10 min at room temperature. Then
the polymer was purified by dissolution-precipitation in methanol-ether
three times, washed with ether, and dried under reduced pressure at
room temperature. Yield 76 mg of R,ω-dithiol-polyHPMA.
Aminolysis of R,ω-di(phenylcarbonothioylthio)-HPMA copolymer-
DOX conjugate was performed similarly as R,ω-di(phenylcarbono-
thioylthio)-polyHPMA.
Preparation of Multiblock PolyHPMA by Chain Extension
with Bis-MAL-dPEG3. R,ω-Dithiol-polyHPMA (70 mg, 2.49 µmol,
Mn 28.1 kDa, PDI 1.08) and bis-MAL-dPEG3 (1.3 mg, 2.49 µmol) at a
molar ratio of 1:1 were dissolved in 160 µL of MeOH, and the reaction
mixture was stirred at room temperature for 24 h. The polymer was purified
by dissolution-precipitation in methanol-acetone three times, washed with
acetone, and dried under reduced pressure. Yield 65 mg of polymer.
Chain extension of R,ω-dithiol-HPMA copolymer-DOX conjugate
(70 mg, 3.00 µmol, Mn 23.3 kDa, PDI 1.19) with bis-MAL-dPEG3
(1.6 mg, 3.06 µmol) was performed similarly. Yield 60 mg of extended
multiblock HPMA copolymer-DOX conjugate.
Enzyme-Catalyzed Degradation of Multiblock PolyHPMA. Pa-
pain and cathepsin B were used for polymer degradation. The activities
of papain and cathepsin B were assessed using the substrate Bz-Phe-
Val-Arg-p-nitroanilide (Bz-Phe-Val-Arg-NAp). The concentration of
the enzyme stock solution in buffer (McIlvaine’s buffer with 2 mM
EDTA, pH 517,18) was measured spectrophotometrically (280 nm, 1
Polymerization. RAFT Polymerization of HPMA Mediated by
Peptide2CTA. HPMA homopolymerizations were performed in metha-
nol at 60 °C using 2,2′-azobisisobutyronitrile (AIBN) as initiator. A
typical procedure was as follows: HPMA (200 mg, 1.4 mmol),
peptide2CTA (7 mg, 5 µmol), and AIBN (0.45 mg, 2.7 µmol) were
dissolved in methanol (1.2 mL). The solution was bubbled with N2 for
30 min, sealed and polymerized at 60 °C for 24 h. After polymerization,
the polymer was purified by dissolution-precipitation in methanol-

Backbone Degradable Multiblock Copolymer Conjugates
Biomacromolecules, Vol. 12, No. 1, 2011 249
Scheme 1. Synthesis of Bifunctional Chain Transfer Agent Containing an Enzyme-Sensitive Peptide Sequence
cm cuvette, using A1% ) 25 and 20 for papain and cathepsin B,
In the coupling reaction, excess (3×) of 4-cyano-4-(phenylcar-
respectively). The concentration of Bz-Phe-Val-Arg-NAp stock solution
in DMF was measured by UV-vis at 316 nm using extinction
coefficient of 1.36 × 104 M-1 cm-1 after 20× dilution with water.
During the measurement, enzyme stock solution was preincubated
in buffer containing 5 mM GSH at 37 °C for 5 min, then Bz-Phe-Val-
Arg-NAp stock solution was added. For papain, the final concentrations
in the incubation mixture were 1.7 × 10-7 M enzyme and 3.69 × 10-4
M p-nitroanilide. The release of p-nitroaniline was measured at 410
nm (using extinction coefficient 8480 M-1 cm-1). At 10 min, the
cleavage was 7.3% of NAp, ∆[NAp]/[enzyme]/min ) 16.6/min. For
cathepsin B, the final concentrations in the incubation mixture were
6.52 × 10-7 M enzyme, 3.34 × 10-4 M p-nitroanilide. The release of
p-nitroaniline was measured at 410 nm (using extinction coefficient
8480 M-1 cm-1). At 10 min, the cleavage was 7.6% of NAp, ∆[NAp]/
[enzyme]/min ) 5.75/min.
bonothioylthio)pentanoic acid and coupling agents were used.
To evaluate the efficiency of the new peptide2CTA, HPMA
polymerization in methanol at 60 °C was studied using 2,2′-
azobisisobutyronitrile (AIBN) as the initiator. The molecular
weight of the polymers, measured by size exclusion chroma-
tography (SEC) and UV-vis (dithiobenzoyl end group deter-
mination at 302 nm), fit well with theoretical values, which
demonstrated the telechelic nature of the polymer and that the
majority of the polymers retained the phenylcarbonothioylthio
group at both ends. Linearity of the pseudo-first-order kinetics
plot (Figure 1A) and the evolution of Mn and Mw/Mn with
conversion (Figure 1B) indicated that HPMA polymerization
was well controlled over molecular weight and polydispersity.
The Mn increased in a linear fashion, with conversion up to
about 40%, and was in agreement with theoretical values.
During RAFT polymerization (Figure 2A) the HPMA mono-
mers incorporated at both dithiobenzoate groups of the
peptide2CTA with identical efficiency. When the final polymer
was incubated with papain, a thiol proteinase with similar
specificity as lysosomal proteinases,20,21 the molecular weight
decreased to half of the original value (Figure 2B).
The degradation of multiblock polyHPMA obtained from thiol-ene
reactions was performed in McIlvaine’s buffer (50 mM citrate/0.1 M
phosphate) at pH 5.0, 37 °C, using papain or cathepsin B. Papain (0.45
mg/mL, 0.1 mL; or cathepsin B, 0.35 mg/mL, 0.1 mL) was mixed with
0.8 mL of buffer containing 5 mM GSH and the mixture was preincubated
for 5 min at 37 °C. Multiblock polyHPMA (260.8 kDa fraction) or
multiblock HPMA copolymer-DOX conjugate (227.7 kDa fraction) in
0.1 mL of buffer containing 4 mg of the polymer was added and incubated
at 37 °C. At scheduled time intervals, 0.1 mL sample was withdrawn and
mixed with 0.4 mL of PBS (pH 7.3) containing 0.05 mL of 1 M sodium
¨
iodoacetate. The sample was analyzed by SEC using an AKTA FPLC
system equipped with miniDAWN TREOS and OptilabEX detectors using
a Superose 6 HR/10/30 column with PBS (pH 7.3) as mobile phase. For
multiblock HPMA copolymer-DOX conjugate, the release of free DOX
was also analyzed by HPLC.
Results and Discussion
To synthesize long-circulating high molecular weight HPMA
copolymer conjugates which are biocompatible and can be
eliminated from the organism, biodegradable bonds should be
incorporated into the polymer backbone. A new bifunctional
RAFT chain transfer agent, NR,Nε-bis(4-cyano-4-(phenylcar-
bonothioylthio)pentanoyl glycylphenylalanylleucylglycyl)lysine
(peptide2CTA) was designed. Using Fmoc-Lys(Fmoc)-OH, two
copies of lysosomally degradable GFLG sequences19 were
introduced at the same time by solid phase peptide synthesis.
Both ends of GFLG sequences were capped with 4-cyano-4-
(phenylcarbonothioylthio)pentanoate (Scheme 1 and Supporting
Information). The long spacer from solid phase peptide synthesis
beads can reduce the steric hindrance, speed up the coupling of
dithiobenzoate chain transfer agent, and reduce the aminolysis of
dithiobenzoate groups. Two copies of GFLG peptide may also
increase the degradation rate during enzyme degradation process.
Different coupling conditions were tested. DIC/HOBt (1/1) cou-
pling method avoided formation of byproduct and gave good yields.
Figure 1. RAFT polymerization of HPMA mediated by peptide2CTA
at 60 °C in methanol: [HPMA]0/[CTA]0 ) 400, [HPMA]0 ) 1 M, [CTA]0/
[AIBN]0 ) 5. (A) Kinetics plot; (B) Evolution of Mn and polydispersity
index vs conversion.

250 Biomacromolecules, Vol. 12, No. 1, 2011
Pan et al.
Figure 2. (A) Polymerization of HPMA in methanol at 60 °C for 24 h mediated by peptide2CTA and AIBN: [HPMA]0/[CTA]0 ) 2000, [HPMA]0 )
1 M, [CTA]0/[AIBN]0 ) 2.5. (B) Following incubation of the polyHPMA with papain, the molecular weight decreased to half of the original value.
Scheme 2. Synthesis of Multiblock HPMA Polymer by Reaction of Telechelic R,ω-Dithiol-polyHPMA with Bismaleimide and Degradation by
Incubation with Cathepsin B or Papain
Telechelic polymers prepared by RAFT polymerization have
been used for attachment of biologically active compounds,
synthesis of block copolymers, and modification of biomaterial
surfaces. Alkyne-azide click reaction or thiol-ene reaction
have been used for the synthesis of multisegment copolymers.
Matyjaszewski and co-workers prepared multiblock copolymers
by Cu+-catalyzed azide-alkyne cycloaddition of R,ω-diazido-
terminated triblock and pentablock copolymers with propargyl
ether.22 The highly efficient thiol-ene reactions23 do not need
a metal catalyst, are compatible with water, and can be
performed under various experimental conditions. Consequently,
this reaction has been used for the synthesis of block polymers,24
dendrimers,25 functional polymers,26 and biomolecules modified
polymers.27 For example, Li et al. prepared telechelic and
modular block copolymers from RAFT polymers by postpoly-
merizationaminolysisandthiol-eneorDiels-Alderreactions.28,29
The thiol-ene chain extension of R,ω-dithiol-polyHPMA was
evaluated. The dithiobenzoate groups of polyHPMA (Mw 30.3
kDa, PDI 1.08) were converted into thiol groups by aminolysis
with n-butylamine in methanol under N2. The telechelic R,ω-
dithiol-polyHPMA was chain extended by reaction with equimo-
lar amount of bis-[1,13-(3-maleimidopropionyl)amido]-4,7,10-
trioxatridecane (Bis-MAL-dPEG3) in methanol at room tem-
perature for 24 h. The product was fractionated by SEC using
preparative Sepharose 6 column and PBS as mobile phase (Scheme
2; Figure 3). Using the peptide2CTA and combination of RAFT
polymerization with thiol-ene reaction, enzymatic degradable
multiblock high molecular weight HPMA copolymer were pre-
pared. However, the chain extension reaction needs further
optimization. Optimization is under way, including aminolysis in
the presence of ene compounds, using protected thiol functionalized
initiators, using templates for chain extension, and so on.
To prove the backbone biodegradability, the multiblock
polyHPMA fraction (Mw ) 260.8 kDa) was incubated with
cathepsin B and papain. Indeed, within 2 h of incubation, the
polymer degraded to short segments with molecular weight 30
kDa, similar to the initial telechelic R,ω-dithiol-polyHPMA
(Scheme 2; Figure 4).
A similar approach was used for synthesizing a copolymer
of HPMA and a drug containing comonomer, MA-GFLG-DOX.
The HPMA copolymer-DOX conjugate was synthesized by
RAFT copolymerization of HPMA and MA-GFLG-DOX (98/2
mol/mol) in methanol using peptide2CTA as the chain transfer
agent and AIBN as the initiator (Scheme 3). HPMA copolymer-
DOX conjugate has a Mw 27.8 kDa and PDI 1.19; content of
doxorubicin, measured by UV-vis spectrometry at 488 nm, in
water using ε488 nm ) 11000 M-1 cm-1, was 7 wt %.
The dithiobenzoyl end groups were aminolyzed to thiol
groups in 10 min. The short incubation time with n-butylamine
Figure 3. FPLC profile of chain extended multiblock polyHPMA. The
dashed traces are the FPLC profiles of fractions obtained by SEC using
a Superose 6 HR16/60 column. The legend indicates the main compo-
nent. The multiblock polyHPMA contains about 17% monoblock, 40%
diblock, 34% tetrablock, 9% octablock, and other multiblock polymers.

Backbone Degradable Multiblock Copolymer Conjugates
Biomacromolecules, Vol. 12, No. 1, 2011 251
Figure 5. UV-vis spectra of R,ω-dithiol-HPMA copolymer-DOX
conjugate at a concentration of 0.5 mg/mL and free doxorubicin.
The R,ω-dithiol-HPMA copolymer-DOX conjugate was
extended by reaction with equimolar bis-MAL-dPEG3 in
methanol at room temperature for 24 h. The product was
fractionated by SEC using preparative Sepharose 6 column and
PBS as mobile phase similar to that of R,ω-dithiol-HPMA
homopolymer (Figure 6).
The backbone degradability and release of free doxorubicin
were measured during incubation of multiblock HPMA copoly-
mer-DOX conjugate (fraction of Mw 227.7 kDa) with cathepsin
B and papain. The backbone degradation was monitored by
SEC, and the release of DOX by HPLC. Most of the multiblock
long chains were cleaved within 30 min; the SEC profiles of
cleaved products, after 4 h of incubation, were similar to initial
copolymer (Figure 7A,B). The amount of released DOX
increased with time (Figure 7C, peak 12.8 min); concomitantly,
the intensity of polymer peak (ca. 13-18 min) decreased. The
identical elution time of the DOX standard with the peaks of
released DOX indicates that aminolysis and chain extension
reaction did not significantly affect the structure of DOX.
Figure 4. FPLC profiles of initial telechelic R,ω-dithiol-polyHPMA (P-
Org), extended multiblock polyHPMA fraction (P-Ext) and degradation
products after incubation with (A) cathepsin B and (B) papain at
different time intervals.
was chosen to avoid structural changes to DOX at basic
conditions. Compared with free DOX, the UV profile of R,ω-
dithiol-HPMA copolymer-DOX conjugate after aminolysis
(Figure 5) shows that no structural changes in DOX occurred.
Scheme 3. Synthesis of Multiblock HPMA Copolymer-DOX Conjugate and Its Degradation by Incubation with Cathepsin B or Papain

252 Biomacromolecules, Vol. 12, No. 1, 2011
Pan et al.
polymers. Postpolymerization aminolysis followed by chain
extension with a bis-maleimide resulted in linear high molecular
weight multiblock HPMA copolymers. These polymers are
enzymatically degradable and the degradation products have
molecular weight distributions below the renal threshold. The
new multiblock HPMA copolymers hold potential as new
carriers of anticancer drugs.
Acknowledgment. The research was supported in part by
NIH grants GM69847, CA51578, and CA132831 and the
University of Utah Research Foundation. We thank Michael
Jacobsen for proofreading.
Figure 6. FPLC profile of chain extended multiblock HPMA copoly-
mer-DOX conjugate. The dashed traces are the FPLC profiles of
fractions obtained by SEC using a Superose 6 HR16/60 column. The
legend indicates the main component. The multiblock copolymer
contains about 17% monoblock, 36% diblock, 36% tetrablock, 11%
octablock, and other multiblock copolymers.
Supporting Information Available. MS, 1H and 13C NMR,
and HPLC profiles for peptide2CTA. This material is available
free of charge via the Internet at http://pubs.acs.org.
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Figure 7. FPLC profiles of initial telechelic HPMA copolymer-DOX
conjugate (PD-Org), extended multiblock HPMA copolymer-DOX con-
jugate (PD-Ext), and degradation products after incubation with (A)
cathepsin B and (B) papain for different time intervals. (C) HPLC profiles
of multiblock HPMA copolymer-DOX conjugate following incubation with
cathepsin B for different time intervals; bottom trace, DOX standard.
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In conclusion, polyHPMA and HPMA copolymer-DOX
conjugates have been synthesized by RAFT polymerization
mediated by a new bifunctional CTA that contains an enzymati-
cally degradable oligopeptide sequence and produces telechelic
BM101254E