Ring-opening polymerization of prodrugs: A versatile approach to prepare well-defined drug-loaded nanoparticles

Article abstract of DOI:10.1002/anie.201409293

The synthesis of polymer-drug conjugates from prodrug monomers consisting of a cyclic polymerizable group that is appended to a drug through a cleavable linker is achieved by organocatalyzed ring-opening polymerization. The monomers polymerize into well-d

Full text of DOI:10.1002/anie.201409293

Angewandte
Chemie
DOI: 10.1002/anie.201409293
Polymer–Drug Conjugates
Ring-Opening Polymerization of Prodrugs: AVersatile Approach to
Prepare Well-Defined Drug-Loaded Nanoparticles**
Jinyao Liu, Wenge Liu, Isaac Weitzhandler, Jayanta Bhattacharyya, Xinghai Li, Jing Wang,
Yizhi Qi, Somnath Bhattacharjee, and Ashutosh Chilkoti*
Abstract: The synthesis of polymer–drug conjugates from
prodrug monomers consisting of a cyclic polymerizable group
that is appended to a drug through a cleavable linker is
achieved by organocatalyzed ring-opening polymerization.
The monomers polymerize into well-defined polymer pro-
drugs that are designed to self-assemble into nanoparticles and
release the drug in response to a physiologically relevant
stimulus. This method is compatible with structurally diverse
drugs and allows different drugs to be copolymerized with
quantitative conversion of the monomers. The drug loading
can be controlled by adjusting the monomer(s)/initiator feed
ratio and drug release can be encoded into the polymer by the
choice of linker. Initiating these monomers from a poly(ethy-
lene glycol) macroinitiator results in amphiphilic diblock
copolymers that spontaneously self-assemble into micelles
with a long plasma circulation, which is useful for systemic
therapy.
yield and enable easy purification of the conjugate; and
4) enable the release of the drug by a biologically relevant
trigger.
Motivated by this rationale, we developed a new method
to synthesize polymer–drug conjugates by living ring-opening
polymerization (ROP) of prodrugs (Scheme 1). This method
M
ost small-molecule drugs utilized in the clinic have poor
bioavailability and suboptimal pharmacokinetics because of
their hydrophobicity and low molecular weight. Polymeric
drug-delivery systems can improve the efficacy of these drugs
by increasing their water solubility, prolonging their circu-
lation time, increasing the amount of drug deposited in the
target tissue, and decreasing their exposure to normal
tissues.[1] Conjugation of hydrophobic drugs to hydrophilic
polymers can address these problems,[2] and is typically
carried out by the separate synthesis of the polymer, drug,
and linker, and sequential conjugation of the three entities to
create the polymer–drug conjugate. This conventional strat-
egy requires multiple reaction steps with limited yield, and
has limited control of the site and degree of drug loading. New
methods are hence needed to synthesize polymer–drug
conjugates. These methods should 1) be compatible with
a structurally diverse set of drugs; 2) enable more than one
drug to be conjugated to the same polymer with tunable
control of the loading of the two drugs; 3) proceed with high
Scheme 1. Schematic illustration of the design and synthesis of
biodegradable polymer prodrugs by living ROP of prodrug monomers,
and self-assembly of the polymer prodrugs into nanoscale micelles.
inverts the conventional approach of conjugating a drug to
a polymer after its synthesis, and instead directly incorporates
the drug during the synthesis of the polymer. These polymer
prodrugs are synthesized from prodrug monomers that consist
of three covalently linked moieties: 1) a cyclic group that can
undergo ROP to give a biodegradable main chain that is
attached to 2) a cleavable linker, which in turn is attached to
3) a drug of interest. Living ROP of prodrug monomers leads
to polymer prodrugs with a biodegradable main chain with
pendant drug molecules that are linked to the main chain
through a cleavable linker. We show that this method is
suitable for the conjugation of structurally diverse drugs.
More than one drug can be copolymerized by this method-
ology and the drug loading and release can be readily
controlled by adjusting the monomer/initiator feed ratio and
by the design of linkers that are cleaved by relevant in vivo
triggers, such as the reductive environment of the cell cytosol.
We further show that by the appropriate choice of an initiator,
in this case poly(ethylene glycol) methyl ether (mPEG), the
second drug-loaded segment can be directly grown from the
mPEG macroinitiator, leading to the formation of an
[*] Dr. J. Liu, Dr. W. Liu, I. Weitzhandler, Dr. J. Bhattacharyya, Dr. X. Li,
Dr. J. Wang, Y. Qi, Dr. S. Bhattacharjee, Dr. A. Chilkoti
Department of Biomedical Engineering, Center for Biologically
Inspired Materials and Material Systems, Duke University
Durham, NC 27708 (USA)
E-mail: chilkoti@duke.edu
[**] We would like to thank Michelle Gignac for the TEM, Yingrong Xu
for the MALDI-TOF MS, and Xiaomeng Wan for the SEC measure-
ments. This work was supported by funding from the NIH (R01-EB
000188 and R01-DK092665) to A.C.
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/anie.201409293.
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amphiphilic diblock copolymer that spontaneously self-
assembles into PEGylated (stealth) micelles with a size and
pharmacokinetics that are suitable for systemic therapy of
solid tumors.
The polymerization of prodrugs was previously achieved
by conventional condensation polymerization.[3] Recently,
living radical and ring-opening metathesis polymerization of
prodrugs have been explored to prepare polymer therapeu-
tics,[4] however, polymer–drug conjugates synthesized by
these methods are non-biodegradable, which limits their
clinical application. We chose organocatalytic ROP[5] for the
synthesis of polymer prodrugs because it is a powerful method
for the synthesis of aliphatic polyesters,[6] polycarbonates,[7]
polypeptides,[8] and polyphosphoesters,[9] which are biode-
gradable. As the starting point for the synthesis of the prodrug
monomer, we chose a commercially available functional ester
intermediate, pentafluorophenyl 5-methyl-2-oxo-1, 3-diox-
ane-5-carboxylate (Carb-C6F5).[10] Our initial choice of anti-
cancer drug was chlorambucil (CL) because the clinical
application of CL is limited by its side effects such as nausea,
myelotoxicity, and neurotoxicity.[11] The prodrug 2 (CarbCL),
consisting of a polymerizable cyclic carbonate linked to an
ethylene glycol linker and CL, was synthesized by the reaction
between hydroxy-functionalized 1 and Carb-C6F5 in tetrahy-
drofuran using CsF as catalyst (Scheme 2a). Details of the
synthesis and characterization of 1 and 2 are described in the
Supporting Information.
We investigated ROP of CarbCL using 1,5,7-triazabicyclo-
[4.4.0]dec-5-ene (TBD) as the organocatalyst and mPEG as
macroinitiator. We chose mPEG as the macroinitiator
because the resulting diblock copolymer, consisting of
mPEG and the polymer prodrug, is amphiphilic and is
likely, we hypothesized, to self-assemble into long circulating
nanoparticles by virtue of PEGsꢀ stealth-like properties.
Trimethylene carbonate (TMC), a commercial available
cyclic carbonate monomer, was used as a co-monomer to
tune the degree of drug loading.
Scheme 2. Detailed synthetic routes of polymerizable prodrugs and
their polymers.
We investigated the copolymerization of CarbCL and
TMC in chloroform at room temperature with different
monomer/initiator feed ratios (Table 1). As shown in Fig-
ure 1a, the ROP of CarbCL and TMC exhibited a linear
evolution of Mn with increasing monomer conversion that is
characteristic of a living polymerization. Gel-permeation
chromatography (GPC) showed monomodal and symmetric
elution peaks for mPEG-poly(TMC-CL)s that exhibited
a clear shift to a higher molecular weight with reaction time
(Figure 1a, inset). In comparison with mPEG, GPC elution
curves of mPEG-poly(TMC-CL)s showed no visible residual
mPEG peak, indicating a high initiation efficacy of the ROP.
Matrix-assisted laser desorption/ionization time-of-flight
(MALDI-TOF) mass spectra of these polymers showed the
absence of a peak at around 5 kDa, suggesting almost
complete consumption of the macroinitiator (Figure S9).
Furthermore, almost quantitative conversion of the mono-
mers was achieved after approximately 17 h reaction time
according to the 1H NMR spectra (Figure S8a and Table S1).
Interestingly, complete consumption of CarbCL was observed
within 10 min, indicating its higher reactivity compared to
TMC. Similar results have been reported for the copolymer-
Table 1: Summary of all polymer prodrugs.[a]
Entry Molar feed ratio DP
Mn
Mw/Mn Dh
[nm]
PDI
M1:M2:M3:M4:I M1:M2:M3:M4 [gmolÀ1
]
1
2
3
4
5
6
7
8
45:5:0:0 :1
43:8:0:0:1
40:10:0:0:1
35:15:0:0:1
40:0:10:0:1
37:0:3:0:1
40:0:0:5:1
43:5:0:3:1
49:4:0:0
48:7:0:0
46:13:0:0
41:16:0:0
45:0:9:0
43:0:2:0
44:0:0:4
47:5:0:2
12100
13400
16300
17400
15800
10600
12400
14000
1.17
1.19
1.23
1.29
1.19
1.27
1.19
1.20
35
0.06
0.06
0.04
0.07
0.04
0.01
0.04
0.05
40
43
49
37
31
30
33
[a] M1, M2, M3, M4, and I are TMC, CarbCL, Carb-O-CPT, Carb-SS-CPT,
and mPEG-5k, respectively. The DP was calculated by 1H NMR
spectroscopy. The Mn and Mw/Mn were obtained from GPC. The Dh and
PDI of the self-assembled micelles were determined by DLS.
ization of TMC with other cyclic carbonate monomers.[12] The
quantitative conversion of the comonomers also facilitated
the purification of mPEG-poly(TMC-CL) with a high yield of
around 85% by precipitation from chloroform into diethyl
ether. The 1H NMR spectrum showed that more than 99% of
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We next investigated the generality of this methodology
by asking the question whether it could be used with other
hydrophobic drugs, for example, bearing hydroxy, amine, or
other functional groups. To answer this question, we chose
camptothecin (CPT), a hydroxy-functionalized anticancer
drug, for the synthesis of a polymer prodrug. CPT is a highly
potent, naturally occurring alkaloid with a wide spectrum of
antitumor activity through the inhibition of topoisomerase I
and HIF-1a.[14] However, its systemic delivery is problematic
because of its low aqueous solubility. As shown in Scheme 2b,
CPT was first activated by triphosgene in the presence of 4-
(dimethylamino)pyridine (DMAP) and then reacted with
excess di(ethylene glycol) to produce intermediate 3. Finally,
prodrug 4 (Carb-O-CPT), consisting of a polymerizable cyclic
carbonate, a di(ethylene glycol) linker, and CPT, was
successfully synthesized by reaction between 3 and Carb-
C6F5 in dimethyl sulfoxide using 1,8-bis(dimethylamino)naph-
thalene (proton sponge) as the catalyst. Copolymerization of
Carb-O-CPTand TMC was performed in chloroform at room
temperature using TBD and mPEG as the catalyst and
macroinitiator, respectively. The conversion of Carb-O-CPT
Figure 1. a) Linearly increasing Mn as a function of monomer conver-
sion for the ROP of CarbCL and TMC using mPEG (Mn of 5 kDa) as
macroinitiator. The inset shows representative GPC curves after
a reaction time of 10 min (red), 1 h (green), and 4 h (pink). b) Plot of
drug loading versus molar feed ratio of CarbCL/mPEG. c) Plot of the
I339.2/I334.9 ratio from pyrene excitation spectra as a function of the
concentration of the polymer prodrugs on a log scale (log C).
d) Representative cryo-TEM image of mPEG-poly(TMC46-CL13) micelles.
1
was nearly complete, as indicated by H NMR spectroscopy
(Table S1, entries 5 and 6). Polymer prodrugs of mPEG-
poly(TMC-CPTO) (the subscript O indicates a chemically
stable ether linker) with a CPT loading from 6.6 wt% to
21 wt% were obtained by increasing the Carb-O-CPT/mPEG
molar feed ratio from 3.0 to 10 (Table 1, entries 5 and 6).
Having synthesized two polymer prodrugs with tunable
loading, we next turned our attention to devising a suitable
release mechanism of the drug from the polymer. To address
this challenge, a reduction-responsive prodrug 6 (Carb-SS-
CPT) was designed and synthesized, as shown in Scheme 2c,
motivated by previous studies on reduction-sensitive polymer
nanoparticles.[15] The synthetic route to 6 is similar to that of
Carb-O-CPT, except that 2,2’-dithiodiethanol was used as
a linker. As expected, mPEG-poly(TMC-CPTSS) (the sub-
script SS indicates a reduction-cleavable disulfide linker) was
synthesized by ROP of Carb-SS-CPT and TMC, using TBD
and mPEG as catalyst and macroinitiator, respectively
(Table 1, entry 7). The drug-release kinetics of mPEG-pol-
y(TMC-CPTSS) under physiological environment were inves-
tigated with or without treatment with glutathione (GSH). As
shown in Figure 2a, rapid drug release was observed for
mPEG-poly(TMC-CPTSS) micelles in the presence of 10 mm
GSH. The cumulative release of CPT reached approximately
75% after 24 h incubation, indicating the reduction-respon-
siveness of mPEG-poly(TMC-CPTSS). Liquid chromatogra-
phy–mass spectrometry analysis of the released products from
mPEG-poly(TMC-CPTSS) showed that the disulfide bond was
cleaved by GSH (Figure S15), which induced the breakdown
of the neighboring carbonate bond to generate free CPT.[16] In
contrast, only around 15% released drug was observed for
mPEG-poly(TMC-CPTSS) micelles when incubated without
GSH. The nonresponsive control, mPEG-poly(TMC-CPTO)
micelles, also displayed slow drug release even upon addition
of 10 mm GSH. Furthermore, very limited drug leakage was
observed by incubating the micelles in serum (Figure S16b).
These release profiles showed that mPEG-poly(TMC-CPTSS)
micelles have good stability under normal physiological
the polymer chains have hydroxy end groups, verifying the
homogeneity of the polymer (Figure S8c). In addition, the
degree of polymerization (DP) of poly(TMC-CL) could be
conveniently adjusted by tuning the monomer/initiator feed
ratio (Table 1, entries 1–4). As shown in Figure 1b, the drug
loading of mPEG-poly(TMC-CL) could be tuned from
10 wt% to around 30 wt% by increasing the CarbCL/
mPEG molar feed ratio from 5.0 to 15. Taken together,
these results confirm that organocatalyzed ROP of prodrugs
enables the facile synthesis of polymer prodrugs with
quantitative monomer conversion and polymerization initia-
tion efficiency, and with an adjustable degree of drug loading.
The amphiphilic nature of these polymer prodrugs also
drives their self-assembly in aqueous media. The critical
micellization concentration (CMC) was determined by using
pyrene as a probe.[13] As shown in Figure 1c, the CMCs of
mPEG-poly(TMC-CL) slightly decreased from 2.5 to
1.1 mgmLÀ1 with an increase in the polycarbonate content
from 58 wt% to 71 wt%. These relatively low CMC values
indicate that the mPEG-poly(TMC-CL) micelles are quite
stable in water. Dynamic light scattering (DLS) showed that
the size of the micelles was tunable by control of the
molecular weight of the hydrophobic polycarbonate segment.
The average hydrodynamic diameter (Dh) of mPEG-poly-
(TMC-CL) micelles increased from 35 to around 50 nm as the
CarbCL/mPEG ratio increased from 5.0 to 15 (Table 1,
entries 1–4). Transmission electron microscopy (TEM)
images further showed that these amphiphilic polymer
prodrugs self-assembled into spherical micelles with a size
that agreed well with the DLS results (Figure 1d, Figure S14
and Table S2).
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We then evaluated the anticancer effects of these polymer
prodrugs in vitro by a cell viability assay in murine C26 colon
and 4T1 breast cancer cell lines; these cell lines were chosen
because they have been reported to be sensitive to CL and
CPT.[18] Both polymer prodrugs exhibited dose-dependent
inhibition of C26 and 4T1 cells. The doses of mPEG-poly-
(TMC-CL) required for 50% cytotoxicity (IC50) against C26
and 4T1 cells were 39 and 1.2 ꢁ 102 mm respectively, which
were about two times higher than those for free CL (Fig-
ure 2b). These results are encouraging because the classical
CL prodrugs often show significantly lower in vitro cytotox-
icity than free CL.[11] The IC50 values of mPEG-poly(TMC-
CPTSS) for C26 and 4T1 cells were 0.32 and 1.4 mm,
respectively, which were much lower than the IC50 values of
mPEG-poly(TMC-CPTO), a polymer prodrug in which the
drug is attached to the polymer through a stable ether linker,
in the same cell lines (Figure 2c). The enhanced cytotoxicity
of mPEG-poly(TMC-CPTSS) compared to mPEG-poly-
(TMC-CPTO) is likely due to its reduction-sensitive linker,
which facilitates CPT release in cells (Figure S17). We note
that extracellular release of camptothecin might occur in cell
culture and in vivo as a result of the presence of thiols
secreted by cells that would lead to cleavage of this nonsteri-
cally hindered disulfide bond between the drug and poly-
mer.[19] Surprisingly, the IC50 value of mPEG-poly(TMC-
CPTSS) against 4T1 cells was even lower than that of free CPT.
The higher cytotoxicity of CPT in the reducible polymer
conjugate compared with the free drug also translated to
a more potent combination polymer prodrug, as the IC50
value of mPEG-poly(TMC-CL-CPTSS) for 4T1 cells was
approximately 0.15 mm, which is 14 times lower than a cocktail
of the free drugs administered at the same drug ratio
(Figure 2d). Together, these in vitro studies clearly demon-
strate that these polymer prodrugs have similar, and in one
case greater, cytotoxicity than the free drug(s), and that this
effect can be modulated by the design of the linker.
To confirm that the base polymer has no intrinsic
cytotoxicity, we cultured C26 and 4T1 cells with mPEG-
poly(TMC)46, a diblock copolymer devoid of drug, and
observed that the polymer exhibited no cytotoxicity even at
a high concentration of up to 1.0 mgmLÀ1 (Figure S19). We
also examined the in vitro degradation of the base polymer in
the presence of lipase, which is known to degrade PTMC and
other aliphatic polycarbonates.[20] The results showed that
about 60% of the carbonate bonds in mPEG-poly(TMC)46
were degraded after six days of incubation with lipase
(Figure 2e), thus demonstrating the biodegradability of the
polycarbonate.
Finally, the in vivo plasma concentration of mPEG-
poly(TMC-CPTSS) was measured as a function of time after
intravenous injection into mice. The data were fitted to a two-
compartment pharmacokinetic model, resulting in a plasma
AUC of 6.1 ꢁ 102 mMh (Figure 2 f). The AUC for mice treated
with the same dose of free CPT was only 71 mMh. This
approximately nine-fold increase in plasma AUC suggests
that these polymer prodrug micelles have a long plasma
circulation by virtue of the stealth-like properties of PEGs
and are likely to preferentially accumulate in solid tumors, as
compared to the free drug.
Figure 2. a) Cumulative drug release from mPEG-poly(TMC-CPTSS)
micelles in PBS at 378C with (&) or without (*) 10 mm GSH.
Nonresponsive mPEG-poly(TMC-CPTO) in the presence of GSH was
used as a control (~). b) Cell viability tested with mPEG-poly(TMC-
CL) (* C26 cells; ! 4T1 cells) or free CL (& C26 cells; ~ 4T1 cells).
c) cell viability treated with mPEG-poly(TMC-CPTSS) (* C26 cells; 3
4T1 cells), mPEG-poly(TMC-PCPTO) (~ C26 cells; " 4T1 cells), or
free CPT (& C26 cells; ! 4T1 cells). d) 4T1 cell viability treated with
mPEG-poly(TMC-CL-CPTSS) (*) or free drugs (&). The cells were
incubated for 72 h and the cell viability (in %) is normalized against
the blank cells in the same experiment. e) Plot of carbonate-bond
content in mPEG-poly(TMC)46 versus incubation time in lipase solu-
tion at 378C. f) Plasma drug concentration of mPEG-poly(TMC-CPTSS)
(*) and free CPT (&) as a function of time after the intravenous
injection in mice.
conditions, but exhibit rapid drug release in a reductive
environment.
To complete our exploration of the flexibility of this
synthetic methodology, we next examined whether it was
amenable to the copolymerization of two different drug-
containing monomers, as many cancers are treated with
a cocktail of different drugs.[17] We hence copolymerized
CarbCL, Carb-SS-CPT, and TMC using TBD and mPEG as
the catalyst and macroinitiator, respectively (Scheme 2d). A
diblock copolymer of mPEG with a random block of
poly(TMC-CL-CPTSS) was easily synthesized with quantita-
tive conversion of the monomers (Table S1, entry 8). The
mPEG-poly(TMC-CL-CPTSS) diblock copolymer contained
a CL/CPT molar ratio of 2.5, close to the CarbCL/Carb-SS-
CPT molar feed ratio (Table 1, entry 8). This suggests that the
total drug loading and ratio of the two drugs of mPEG-
poly(TMC-CL-CPTSS) can be controlled by adjusting the
molar feed ratio of the two drug-containing comonomers.
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Received: September 22, 2014
Revised: October 21, 2014
Published online: && &&, &&&&
Keywords: cancer therapy · nanoparticles · polymer–
.
drug conjugates · polymerizable prodrugs ·
ring-opening polymerization
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Angew. Chem. Int. Ed. 2014, 53, 1 – 6
ꢀ 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.angewandte.org
5
These are not the final page numbers!

.
Angewandte
Communications
Communications
Polymer–Drug Conjugates
J. Liu, W. Liu, I. Weitzhandler,
J. Bhattacharyya, X. Li, J. Wang, Y. Qi,
S. Bhattacharjee,
A. Chilkoti*
&&&& — &&&&
Conjugates: Biodegradable polymer–
drug conjugates were synthesized
through living ring-opening polymeri-
zation of prodrug monomers consisting
of a cyclic polymerizable group that is
attached to a drug through a cleavable
linker. The polymer–drug conjugates are
designed to self-assemble into nanopar-
ticles and release the drug in response to
a physiologically relevant stimulus.
Ring-Opening Polymerization of
Prodrugs: A Versatile Approach to
Prepare Well-Defined Drug-Loaded
Nanoparticles
6
www.angewandte.org
ꢀ 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Angew. Chem. Int. Ed. 2014, 53, 1 – 6
These are not the final page numbers!