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FEATURE ARTICLE

Cite this: Chem. Commun., 2013,


49, 2082

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Acid-degradable polymers for drug delivery: a decade


of innovation
Sandra Binauld and Martina H. Stenzel*
Polymers that start degrading under acidic conditions are increasingly investigated as a pathway to
trigger the release of drugs once the drug carrier reached the slightly acidic tumour environment or
after the drug carrier has been taken up by cells, resulting in the localization of the polymer in the

Received 11th September 2012,


Accepted 7th December 2012
DOI: 10.1039/c2cc36589h

acidic endosomes and lysosomes. The advances in the design of acid-degradable polymers and drug
delivery systems have been summarized and discussed in this review article. Various acid-labile groups
such as acetals, orthoester, hydrazones, imines and cis-aconityl, that can undergo cleavage in slightly
acidic conditions, have been employed to create polymer architectures or polymerdrug conjugates that

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can degrade under lysosomal and endosomal conditions, triggering the fast release of drugs or DNA.

Introduction
Polymers are a popular choice for the design of drug delivery
carriers. The use of polymer therapeutics has evolved into a
broad discipline, employing a wide range of architectures either
at the macroscale (gels and hydrogels) or nanoscale (polymer
drug conjugates, polymeric micelles, nanogels and cross-linked
particles, polyplexes for DNA delivery, etc.). Particularly, the
development of multifunctional nanoscale devices has drawn a
lot of attention. The versatility of modern synthetic chemistry
as well as recent developments of very ecient click reaction
enabled the careful design of polymer-based therapeutic agents
with engineered characteristics such as colloidal stability, tunable sizes with narrow distribution, protection of drugs during
circulation, and transportation to targeted organ or tissue.
In summary, controlled release of encapsulated therapeutics
is an active research field. A few strategies have been developed
including bioconjugation with cell-targeting biomolecules for
specific delivery and the use of stimuli-responsive polymers to
create intelligent polymer therapeutics.
Stimuli-responsive polymers are defined as polymers that
undergo relatively large and abrupt, physical or chemical
changes in response to small external changes in the environmental conditions. These stimuli could be classified as either
physical (temperature, electric or magnetic fields, mechanical
stress) or (bio)chemical (pH, ionic strength and chemical
agents) stimuli. The response of a polymer can be defined
in various ways, from a reversible change in their chain conformation or their degree of intermolecular association to more
Centre for Advanced Macromolecular Design, (CAMD), The University of
New South Wales, Sydney NSW 2052, Australia. E-mail: m.stenzel@unsw.edu.au

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Chem. Commun., 2013, 49, 2082--2102

dramatic alterations such as degradation by irreversible bond


breakage. Some systems have been developed to combine two
or more stimuli-responsive mechanisms into one polymer
system. This behaviour can be utilised for the preparation of
so-called smart drug delivery systems, which aim to mimic
biological response behaviour. Recent advances of stimuliresponsive polymers in bio-related applications have been
thoroughly reviewed elsewhere.13
Among the various existing stimuli-responsive systems,
pH-sensitive polymers have been widely studied for drug delivery
applications. Indeed, it has been shown that the environment
in tumour tissue is often 0.51.0 pH units lower than in normal
tissue.4 Moreover, a larger pH shift from 7.27.4 in the blood or
extracellular spaces to 4.06.5 in the various intracellular
compartments takes place during cellular uptake that can be
used for intracellular drug delivery.
These changes in the external pH can be exploited by
pH-responsive polymers which respond to pH changes with
an altered solubility, volume, configuration or conformation.
They can be divided in two categories: polymers with ionizable
groups in their backbone and those containing a degradable
linker. The first category includes polyelectrolytes containing
weak acids or bases like carboxylic acids, phosphoric acid and
amines, respectively, that will exhibit a change in the ionisation
state upon pH variation. This will lead to a reversible conformational change for the soluble polymers and a change in the
swelling behaviour for cross-linked systems. These systems
have already been thoroughly discussed,5,6 and are beyond
the scope of the present review. The second category is
composed of polymers containing an acid-degradable linkage.
The acidic conditions in tumour tissue, endosomes, and lysosomes can induce the cleavage of such systems. Coupling drugs
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Feature Article
to suitable carriers through acid-sensitive bonds is one way of
ensuring the triggered release of the drug in an acidic environment. These acid-degradable systems have been quite popular
for the design of smart drug delivery devices in the past decade,
and surprisingly poorly reviewed except some reports on polymeric prodrugs79 or micellar systems.10,11 Moreover, the multitude of names or keywords coined for acid-degradable,
including acid-labile, acid-cleavable, acid-sensitive,
pH-dependant, makes them dicult to track in the literature.
The aim of this review is to highlight the dierent strategies
based on acid-degradable polymeric systems that have been
developed in the past decade and their application. Acidsensitive polymers have been utilized in various drug delivery
devices such as degradable linear polymers and hydrogels,
cross-linked particles, self-assembled particles, polymerdrug
conjugates, and polyplexes (Fig. 1). For each strategy, the
performances of various systems such as degradation time or
drug release will be compared.

Common acid-degradable bonds


A range of chemical bonds are known to be instable in strong
acid or basic solution. This is the case for the various protective
groups commonly used in organic synthesis. However, only a
few of them show an enhanced degradation or hydrolysis in
the presence of slightly acidic media, while being stable at
neutral pH. This unique property, determined by the chemical

Sandra
Binauld
studied
materials science and polymer
chemistry at the National
Institute of Applied Sciences
of Lyon (INSA), a French
engineering school. She received
her PhD degree in 2009 from
the
University
of
Lyon
(France), after working with
Prof. E. Drockenmuller and
Prof. E. Fleury on the
elaboration of macromolecular
objects using Click Chemistry.
Sandra Binauld
In 2010, she moved to Australia
to work as a research associate at the Center for Advanced
Molecular Design (University of New South Wales, Sydney)
under the supervision of Prof. M. Stenzel, where she developed
smart drug delivery systems using RAFT polymerization. In 2012,
she joined Prof. B. Charleuxs group (C2P2, Lyon) as a casual
lecturer, and her current research focuses on the development of
nanomaterials using MADIX polymerization induced self-assembly
in aqueous media.

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ChemComm
structure of the bond, makes them candidates of choice for the
preparation of acid-degradable drug delivery systems. The acidsensitive linkers most commonly employed in the literature
include orthoester, acetal, hydrazone, imine, cis-aconytil and
trityl bonds. Their chemical structure and degradation products are given in Fig. 2. The orthoester bond is a functional
group containing three alkoxy groups attached to one carbon
atom that can be prepared by the reaction of nitriles with
alcohols under acid catalysis. They are readily hydrolyzed in
mild aqueous acid to form esters, and therefore usually used as
protecting groups for esters. The acetal bond is a molecule with
two single-bonded oxygen atoms attached to the same carbon
atom (or two carbon-bonded R groups in the case of ketal). In
an acidic solution, one oxygen of the acetal group is protonated,
which activates the neighbouring carbon. This facilitates the
attack of water, resulting in the cleavage of the acetal to the
appropriate aldehyde and alcohol. An imine is a functional
group containing a carbonnitrogen double bond, with the
nitrogen attached to a hydrogen atom or an organic group. If
this group is not a hydrogen atom, then the compound is more
stable and defined as a Schi base. They are obtained by
reaction of an aldehyde or ketone with a primary amine. When
an amine group is attached to the nitrogen, the imine is called
hydrazone. Hydrazones are usually formed by the reaction of
hydrazine on ketones or aldehydes. The cis-aconityl linker is a
derivate of natural aconitic acid that has a carboxylic acid (C-4)
in cis-position to a hydrolytic bond (C-1). This linker undergoes

Martina
Stenzel
studied
chemistry at the University of
Bayreuth, Germany, before
completing her PhD in 1999 at
the
Institute
of
Applied
Macromolecular
Chemistry,
University
of
Stuttgart,
Germany. She started working
as a DAAD postdoctoral fellow
at the University of New South
Wales
(UNSW),
Sydney,
Australia, where she currently
holds the position of full
Martina H. Stenzel
professor and ARC Future
Fellow. Her research interest encompasses the synthesis of
functional polymers with complex architectures such as
glycopolymers and other polymers for biomedical applications,
especially polymers with in-build metal complexes for the delivery
of metal-based anti-cancer drugs. Martina Stenzel has authored
more than 190 peer reviewed papers and 7 book chapters mainly
on RAFT polymerization. She is currently the Honorary Secretary
of the Royal Australian Chemical Institute and a member of the
Australian Research Council (ARC) college of experts. She is an
editor of the Australian Journal of Chemistry and also serves on
several editorial boards (Polymer, Progress in Polymer Science,
Macromolecules, Biomacromolecules, ACS MacroLetters). She
received a range of awards including the 2011 Le Fe`vre Memorial
Prize of the Australian Academy of Science.
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Fig. 1

Feature Article

Examples of acid-degradable systems for drug delivery.

processed into microparticles to branched and crosslinked


polymer with a focus on gel and crosslinked microparticles.

Linear bioerodible polymers and their use in


microparticles preparation

Fig. 2

Examples of acid-degradable bonds and their degradation products.

an intramolecular assisted C-4 acid-catalysed hydrolysis at the


C-1 bond, due to the proximity of the pendent carboxylic acid.

Degradable linear polymers and hydrogels


Biomaterial design often relies on the degradation of the
polymer to enable clearance of the material. In the following,
we will discuss polymers with incorporated acid-cleavable bonds.
The list ranges from simple linear polymers which have been
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Among the bioerodible linear polymers, polyorthoesters have


the longest history since they have been under investigation
since 1970. These polymers have already been extensively
reviewed by Heller et al.12,13 and will not be further discussed
in this article. An increasing interest has been shown in the
past few years for the design of linear polyacetals.1424 Heller
et al. first reported the synthesis of linear and cross-linked
polyacetal by condensation of polyols with divinyl ethers.14 This
reaction has been applied more recently by Tomlinson et al. to the
synthesis of water-soluble, biocompatible, amino-functionalized
polyacetals, suitable for drug conjugation (Fig. 3, 1).15 In vitro
studies showed that the polymers and their degradation products were nontoxic and the polyacetals showed no preferential
accumulation in the major organs. In the same group, Vicent
et al. used a similar system to incorporate a drug with a bishydroxyl functionality (non-steroidal oestrogen diethylstilboestrol
(DES)) into the polymer backbone (Fig. 3, 2).16 These bioresponsive DES-polyacetals tert-polymers are the first watersoluble anticancer polymeric drugs designed for acidic
pH-triggered release. Schacht et al. from Hellers group also
reported the synthesis of graft copolymers 3 comprised of a
polyacetal backbone with pendant poly(ethylene glycol) sidechains. These graft polymers were used to prepare a series of
thermogels with a lower critical solution temperature (LCST)
between 25 and 60 1C.17 Fine-tuning of the LCST was achieved
by carefully adjusting the hydrophilichydrophobic balance by
altering the PEG content. The LCST could then be targeted
towards the application, similar to PNIPAAm polymers.25 More
rapidly eroding thermogels were prepared by replacing the
polyacetal backbone with a poly(ortho ester) backbone (Fig. 3, 4).
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Fig. 3

ChemComm

Linear polymers with degradable functional groups in the main chain.

Using a similar coupling reaction, Garripelli et al. reported the


synthesis of a novel thermo-sensitive multiblock copolymer 5,
which was obtained from a Pluronic triblock copolymer and
di-(ethylene glycol) divinyl ether.18 The aqueous solutions of
these polymers underwent gelation at body temperature, allowing
the release of FITCdextran (40 kDa) in a pH-dependent fashion.
In a dierent way, Jain et al. prepared poly(amidoamine)s by
the stepwise polyaddition of primary or secondary amines
to bis(acrylamide) monomers containing acid-degradable
acetaldehyde acetal and dimethyl ketal linkages (Fig. 3, 6).19
The molecular weights were rather low since the bis(acrylamide)
monomers degraded via hydrolysis of the acid-degradable functional group during polymerization. The rate of degradation was
influenced by the type of acetal structure used within the backbones (Fig. 3, 6).
Linear polyacetals have also been used by Murthys and
chets groups to obtain degradable microparticles for drug
Fre
delivery. Specifically, Heerman and Murthy formulated
dexamethasone-loaded polyketal nanoparticles containing poly(1,4-phenyleneacetone dimethyleneketal) (PPADK) (Fig. 4, 7).21
In the same group, Khaja et al. used the acyclic diene metathesis
(ADMET) polymerization to obtain a variety of polyacetals and
polyketals which could be processed into microparticles
(Fig. 4, 8).22 A library of polyurethanes and polyureas-based
polyacetals (Fig. 4, 9) has been prepared by Frechets group by
step-growth polymerization of bis(p-nitrophenyl carbamate/
carbonate) or diisocyanate monomers with an acid-degradable,
ketal-containing diamine.23 Interestingly, degradation studies
showed that the hydrolysis kinetics is closely related to the
hydrophobicity of the starting polymer. To further explore these
acid-degradable materials, microparticles were prepared from
polymer 10 to evaluate their potential as carriers for proteinbased vaccines. It has been demonstrated that particles made
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Fig. 4 Linear acid


microparticles.

degradable polymer used for the

preparation of

from polymer 10 are more eective in generating an immune


response compared to free protein and analogous particles
prepared from a slower degrading polymer.24
Even though the large majority of studies on linear aciddegradable polymers were based on polyacetal or polyorthoester
backbones, a few examples explored other types of acid-degradable
bonds. For instance, Zhou et al. synthesized pH-sensitive
multifunctional polyurethane micelle drug carriers based on
macrodiol containing acid-cleavable hydrazone linkers.26,27
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Also linear polyhydrazone28 and water soluble poly(cis-aconityl)29
have barely been investigated.

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Hydrogels and branched polymers


Hydrogels are usually formed from a polymer chain network,
creating a colloidal gel containing often more than 99% water.
They combine the advantages of high absorbency and permeability. The medical and pharmaceutical uses of hydrogels
include wound dressing, skin grafts, oxygen-permeable contact
lenses and delivery of drugs. For the latter application, the
synthesis of biodegradable materials, which disintegrate into
non-toxic degradation products, is highly desirable. Once again
the development of acetal or ketal-based polymer networks has
led the way. Several studies have focused on the development of
cyclic acetal biomaterials, that degrade into non-acidic products, terminated with diol and carbonyl end groups.30 More
recently, Sui et al. prepared an acetal-based polymer network by
combining reversible additionfragmentation chain-transfer
polymerization (RAFT) with addition reactions by reacting the
hydroxyl pendant groups of the polymer with 1,4-cyclohexanedimethanol divinyl ether.31 Chatterjee and Ramakrishna
reported the synthesis of hyperbranched polyacetals via a melt
transacetalization polymerization process of an AB2 monomer
bearing a single hydroxyl group and a dialkyl acetal.32 The
bulk degradation rates of these hyperbranched polyacetals
under mildly acidic pH (pH = 4) revealed the strong dependence
of the degradation rates on the nature of peripheral alkyl
substituents.
The chemistry of hydrazone conjugation via aldehyde
hydrazide cross-linking is also of great interest for the preparation of hydrogels. This is a straightforward and high yielding
reaction, which is well suited for drug design and delivery due
to the relevant pH range in which these materials degrade.
Particularly, this reaction can be performed in vivo, allowing the
preparation of injectable hydrogels, and does not have any
adverse tissue eects.33,34 These novel biomaterials can thus be
employed for controlled release of therapeutic agents at wound
sites. An array of studies has focused on the preparation of
hyaluronic acid hydrogels via hydrazone cross-linking.3542 This
cross-linking strategy has been chosen because the modification of HA to hydrazide and aldehyde functionalities is straightforward, and the reaction is very ecient, with gelation usually
occurring within a minute after mixing of the precursors. It has
been proven that the hydrazone hydrogels remain more stable
at physiological pH compared to their imine analogues.35
With the help of microfluidic technology, carbohydrate
hydrazone-cross-linked gels can also be obtained at the microscale for the design of smart microgel particles with sizes
ranging between E40 and 100 mm.43 Moreover, injectable
hydrogels based on a mixture of natural and thermo-sensitive
synthetic polymers were recently obtained by Patenaude and
Hoare.44 A series of synthetic oligomers of poly(N-isopropylacrylamide) (PNIPAM) and carbohydrate polymers (including
hyaluronic acid, carboxymethyl cellulose, dextran, and methylcellulose) were functionalized with hydrazide or aldehyde functional groups and mixed to create hydrazone-cross-linked hydrogels,
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Feature Article
which gelled in situ. Based on the same idea, analogues of
thermoresponsive PNIPAM hydrogels have been designed by
mixing aldehyde and hydrazide-functionalized PNIPAM oligomers. The hydrogels exhibit the same thermal swelling
deswelling responses as conventional PNIPAM hydrogels, but
can be degraded back into the reactive polymer gel precursors
via an acid-catalyzed hydrolysis process.45
However, only a few reports deal with the use of these aciddegradable hydrogels as drug carriers. Tian et al. developed a
new antibody (IgG) releasing system by covalently attaching IgG
to the biodegradable hyaluronic acid hydrogel using the hydrolytically unstable hydrazone linkage with the aim to deliver the
antibody to the injured brain.38 In pH 5 and 6 buer solution,
most of antibody (6080%) was released from the hydrogel
within 8 and 70 h, respectively, whereas the release was much
slower in pH 7.4 buer solution (over 400 h).

Cross-linked particles via copolymerization


with acid-degradable divinyl monomers or
via post-crosslinking
An omnipresent challenge in the field of drug delivery is the
development of ecient spherical delivery vehicles. Their ideal
characteristics would include a small tuneable size, a high
loading capacity of the cargo, good biocompatibility and a long
circulation time. Regarding this last aspect, it is important that
delivery vehicles can be responsive to their local environment
in order to retain their cargo until they reach their target. The
introduction of a stimuli-responsive cross-linker during the
preparation of the particles can trigger the disintegration of
the drug carrier and the release of the drug once the particle
reached its target.
The development of cross-linked microparticles using an
acetal crosslinker has been introduced and thoroughly studied
chet group since 2002. Following this trend, many
by the Fre
studies have focused on the design of acid-degradable nanogels, nanocapsules and cross-linked micelles. As a result, a
variety of cross-linkers with various functionalities and degradation profiles has been synthesized. The eciency of these
degradable systems in terms of drug release has been summarized in Table 1 and the corresponding crosslinkers are
displayed in Fig. 5 and 6.

Table 1

Hydrolysis half-life of acetal cross-linkers 1120 (Fig. 5 and 6)

Half-life time
Cross-linker

Ref.

pH 5.5 (min)

pH 7.4 (h)

11a
11b
12
13
14
15
16
17
18
19
20

46
47
48
49
50
51
51
51
23
52
53

5.5
5.5
1.6
n.a.
60
20
30
30
60
n.a.
14

24
24
6.8
n.a.
>20
>20
>20
>20
25
n.a.
29

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Fig. 5

Fig. 6

ChemComm

Acetal cross-linkers used for the synthesis of acid-cleavable particles by inverse-emulsion polymerisation.

Acetal cross-linkers used for micelles stabilization.

Microgels/microparticles by inverse microemulsion


polymerization
The first example of acetal cross-linked, protein loaded hydrogels
microparticles, prepared by inverse microemulsion polymerizachet group in 2002.46 It involved
tion, has been described by the Fre
a benzaldehyde acetal cross-linker, which had the added advantage that the acid lability can be manipulated by introducing
substituents in the para position of the phenyl group.
It was therefore possible to engineer the hydrolysis kinetics of
these gels to suit the application. Therefore, the bisacrylamide
acetal cross-linker (11a) with a p-methoxy substituent was chosen
as the acid degradable linkage to ensure that the resulting
microgels hydrolyzed rapidly within the pH 5.0 environment that
is encountered in phagolysosomes. Indeed, hydrolysis was proven
to be extremely rapid at pH 5.0, with a half-life of 5.5 min, whereas
the system remained stable at pH 7.4, with a half-life of 24 h.
A more hydrophilic version of this cross-linker, containing a
hydrophilic triglyme moiety (11b), was synthesized by the same
group, resulting in similar degradation times.47 The triethylene
glycol pendant group incorporated in 11b increased the water
solubility, making it much more hydrophilic and therefore
more suitable for use in inverse emulsion polymerization than
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its para-methoxy substituted analogue, 11a. It was demonstrated


that the rate of protein release from these microgels was
pH-sensitive, with 80% release of encapsulated ovalbumin observed
in 6 h at pH 5.0, but only 10% at pH 7.4. This system was then used
to encapsulate plasmid DNA.54 Retention of the DNA payload at
physiological pH was achieved while the cargo was completely
released under acidic conditions at lysosomal pH. By trapping the
plasmid DNA within the cross-linked microparticle, enzymatic
degradation was prevented when exposed to serum nucleases.
The system was then optimised and evaluated by in vivo experiments. The ability of the protein-loaded particles to provide immunity against tumours in mice was investigated using the ovalbumin
model.55 The same cross-linker was also used to prepare aciddegradable cationic nanoparticles as carriers of a model antigenovalbumin for protein-based vaccine development.56
In 2004, Kwon et al. described the use of new functional
aliphatic monomers and cross-linkers for the preparation of
degradable microparticles with improved properties including
increased biocompatibility and faster hydrolysis.48 Since the
aromatic aldehyde liberated after hydrolysis of cross-linker 11
might react with intracellular proteins, they developed the alternative acid-labile cross-linker 12 that produces acetone as degradation product, a relatively nontoxic metabolic intermediate of
fatty acid oxidization. In addition, an acetal amine-functionalized
acrylate monomer was also designed for its ability to generate
small molecules upon acid hydrolysis. Cross-linker 12 was
hydrolyzed approximately 250 times faster at pH 5.0 than at
the physiological pH of 7.4 i.e. three times faster than the
benzylidene acetal 11. The pH-sensitive polyacrylamide particles were furthermore functionalized with the cell-penetrating
peptide polyarginine to promote cellular uptake.57
Meanwhile, Shi et al. used crosslinker 13 to obtain acid
labile poly(N-vinylformamide) (PNVF) nanogels by inverse
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microemulsion polymerization.58 Acid-labile PNVF nanogels showed
increased stability at neutral pH (pH 7.4; t1/2 > 56 h) compared to
rapid dissolution observed at lower pH (pH 4.7; t1/2 B 10 min).
Using dispersion polymerization and RAFT polymerization, Chan
et al. employed a new bisacrylate acetal cross-linker 14 to create
acid-cleavable coreshell nanoparticles for the delivery of hydrophobic drugs.50 The particles were cleaved in a pH dependent
manner similar to the acid-labile hydrolysis behaviour of the
cross-linker itself. The same group also investigated the degradation of a range of bisacrylate acetal cross-linkers (1417) of
dierent polarities, which were used to prepare PHEMA nanogels
particles.51 Comparison of the hydrolysis half-life of the crosslinkers 14 and 16 leads to the conclusion that the presence of the
hydroxyl group as a p-substituent group on the benzene ring
makes the crosslinker more susceptible to acid-catalyzed hydrolysis compared to the cross-linker with the methoxy group.
Influence of the polarity on the degradation was also discussed.
Nanocapsules
chets group prepared
More recently, Broaders et al. from Fre
acid-degradable microcapsules via interfacial polymerization.
The polyamide was formed by the reaction of acid chlorides
with ketal-containing diamine 18 along the wateroil interface.
Rapid degradation into biocompatible products and complete
release of the encapsulated material was triggered upon exposure to an acidic environment.59 In vitro studies showed that
equivalent non-degradable capsules do not lead to significant
cell death regardless of the toxicity of the encapsulant, whereas
acid-degradable capsules lead to high cytotoxicity when loaded
with paclitaxel. The release of the drug, thus the cytotoxic eect,
was only made possible by the ecient uptake of the nanocapsules by cells. Shi and Berkland reported the synthesis of acidlabile poly(N-vinylformamide) (PNVF) nanocapsules by free radical
polymerization of N-vinylformamide in the presence of crosslinker 13 on the surface of silica nanoparticles.60 The formamide
side group of PNVF was then hydrolyzed by extended exposure to
sodium hydroxide to produce polyvinylamine (PVAm) micro- and
nanocapsules. Both capsule types underwent an increasing dissolution rate as the pH decreased. The extent of cross-linking was
observed to influence the degradation rate of the capsules. For
example, doubling the molar content of crosslinker from 6.5% to
13.2% resulted in a 3-fold increase in capsule half-life at pH 5.0
and produced more gradual degradation kinetics.
Cross-linked micelles
Another popular drug delivery technique involves the use of
micellar systems, which are obtained by self-assembly of amphiphilic copolymers. It is well-accepted that the micellar carriers
potentially allow passive targeting of drugs to tumours via
enhanced permeation and retention eect. However, the one
major drawback is the premature dissociation of these structures
in in vivo conditions due to the high dilution upon exposure to
large volumes of body fluids. To overcome this problem, micelles
are often stabilized by crosslinking. Crosslinking strategies include
core or shell cross-linking, but also crosslinking on the interface
between both blocks is known.61 The use of a stimuli-sensitive
cross-linker is particularly interesting since the disassembly of the
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micelles and subsequent release of the drug can be triggered by
external stimuli, such as pH. Several studies reported the synthesis
of core-cross-linked micelles using an acetal cross-linker in conjunction with RAFT polymerisation.62 Two approaches were investigated: a radical crosslinking process where the acid-degradable
divinyl monomer was copolymerised inside the core of the micelle
to stabilize the structure63 or a post-crosslinking approach, where
the integrity of the micelle was enhanced by employing a difunctional crosslinker.
Zhang et al. used RAFT polymerization to chain-extend a
thermoresponsive block copolymer, poly(acryloyl glucosamine)block-poly(N-isopropylacrylamide), with the commercial orthoestertype cross-linking agent 21 after the block copolymer has been
self-assembled into micelles.64 Chan et al. used a similar approach
and polymerized an acid-cleavable diacrylate cross-linker 14 inside
the core region of the micelles, leading to the chain extension of
the living RAFT end groups. The core cross-linking process was
found to have a minor eect on the original size of the micelle and
the core-segment polarity.65 The same technique was employed by
Bhuchar et al. to generate core cross-linked micelles in a one-pot
process by RAFT polymerization in the presence of a shorter
derivative of the di-methacrylate acetal cross-linker.66
An alternative approach is the stabilisation of micelles using an
acid-degradable difunctional crosslinker. Duong et al. eciently
reacted diamino cross-linker 18 inside a pentafluorophenyl-bearing
micelle core, which was formed by self-assembly of PEB-b-P(vinyl
benzylchloride-co-pentafluorophenyl acrylate) diblock copolymer.67
The same cross-linker was used by Huynh et al. in a reaction
between pendant activated esters at the nexus between micelle core
and shell. The micelle was obtained from a triblock copolymer
of poly(oligo(ethylene glycol)methylether methacrylate)-b-poly(N-hydroxysuccinic methacrylate)-b-poly(1,1-di-tert-butyl-3-(2(methacryloyloxy)ethyl) butane-1,1,3-tricarboxylate), which was
designed for the improved delivery of cisplatin. The acid
degradable crosslinker was directly compared to a non-degradable
crosslinker in their cytotoxicity against cancer cell lines, showing
that the degradable micelle had a much faster mode of action.68
Shell cross-linking is an alternative possibility to enhance micellar
stability against micelle-destabilizing conditions, but it can also
delay the drug release at extracellular pH values. Lee et al. and
Li et al. used diamino cross-linkers 18 and 20 to perform shell
cross-linking of acid-bearing block copolymers.53,69 Tappingmode atomic force microscopy (AFM) was applied as an interesting alternative technology to demonstrate the disassembly of
the acid-labile cross-linked knedel-like (SCK) nanoparticles.53
Finally, cross-linking of some alkynylated micelles was readily
achieved by the alkyneazide click reaction using the azidefunctionalized cross-linker 19.52

pH-induced transformation of
self-assembled systems
Destabilizing micellar systems
One approach to the development of acid-sensitive drug
delivery systems has been to incorporate titratable groups such
as amines and carboxylic acids into the copolymer backbone,
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Table 2

Composition and release eciency of destabilizing micellar systems

Structure/
ref.

Shell

Core

Acid-sensitive group

Drug

22

PEG

Poly(aspartic acid)

Nile red

n.a.

60

2372

PEGdendritic
polyester
PEG
PNIPAM

Hydrophobic
acetals
P(tNEA)
P(OPD-co-CL)

Trimethoxybenzylidene
acetal
Trimethoxybenzylidene
acetal
Cyclic orthoester
Hydrazone

DOX

12

92

80

Nile red
5-Fu

5
9/20

5
48

100
88

0
76

73

24
2579
a

Time
(h)

Releasea (%)

Drug
loading

10

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pH 5

pH 7.4

Values estimated from in vitro release profiles at both pH.

thus altering the solubility of the polymer upon protonation,


resulting in the disruption of the micelle. This approach has
already been reviewed elsewhere and will not be discussed in
the present article.5,70 Another way to induce a pH-dependant
drug release is to incorporate acid-degradable linkages into the
copolymer, causing a structural change of such magnitude that
micellar integrity is lost (Table 2). This approach, based on the
attachment of hydrophobic groups to one block of an amphiphilic copolymer via an acid sensitive linkage, was first proposed
chet and Gillies, who developed the acid sensitive copolymer
by Fre
micelles based on the PEG-b-P(aspartic acid) copolymer 22
(Fig. 7).10 After hydrolysis of the acetal, the hydrophobic block
takes on hydrophilic properties, thus destabilizing the micelle
and enabling fast release of the drug. In this study, cyclic
benzylidene acetals were used as the acid-sensitive linkage,

Fig. 7

because they combine a hydrophobic aromatic ring that will


contribute to micelle formation with a high rate of hydrolysis at
slightly acidic pH. The fast rate of hydrolysis was achieved by
the presence of electron-donating methoxy groups in the ortho
and para positions. Hydrolysis of the acetal results in a more
polar diol moiety, leading to disruption of the micellar assembly and the triggered release of the micellar content. The same
team further developed this system using a similar concept.
PEO with a dendritic end-functionality was functionalized at
the periphery with hydrophobic groups using acid-sensitive
acetal linkages (Fig. 7, 23).71 Upon hydrolysis and the loss of
the hydrophobic groups, the core-forming block becomes
hydrophilic, thus destabilizing the micelle and enabling escape
of the drug from the micelles. This system was tested for selective
release of DOX at acidic pH.72 Based on the same approach,

Structures of copolymers forming destabilizing micellar systems.

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Huang et al. chose to use poly(acrylamide) with pendent


orthoester as hydrophobic block. The orthoester-containing
monomer, trans-N-(2-ethoxy-1,3-dioxan-5-yl)acrylamide (tNEA),
was used to obtain a block copolymer by ATRP that is both
pH-sensitive and thermoresponsive (Fig. 7, 24).73 Upon heating
above the critical aggregation temperature, the polymer underwent a phase transition and formed polymeric micelles with PEG
as the shell and PtNEA block as the core. Dissociation of the
micelles and the subsequent release of Nile Red were induced by
the acid-triggered hydrolysis of the orthoester groups. The same
group investigated other poly(meth)acrylamide derivatives with
pendent six-member cyclic orthoester groups, varying the alkyl
substitutes and the orthoester stereochemical structures.7476
Both thermo-sensitive properties and acid-triggered hydrolysis
behaviours of the polymers were found to be dependent on the
polymer structures. The combination of such acid-sensitive and
thermoresponsive properties was also investigated by other
research groups.77,78 A dierent strategy was developed by He
et al., who grafted thermoresponsive PNIPAM chains to a poly(2-oxepane-1,5-dione-co-e-caprolactone) (P(OPD-co-CL, 25)) via an
acid-sensitive hydrazone linker. As a result, dual-responsive
coreshell micelles with a PNIPAM shell were formed.79 The
graft polymeric micelles exhibited thermo-triggered decelerated
release at pH 7.4, and pH-triggered accelerated release at 25 1C.
An interesting approach was recently presented by Deng et al.
Polyvinylalcohol (PVA) was modified with PEG using an amide
functionality and with adamantane (Ad) pendant groups using a
cyclic acetal linker.80 Acid-catalyzed cleavage of those acetallinked Ad groups from the polymer results in destabilization of
the polymer micelles and release of its hydrophobic cargo.

Table 3

A very dierent type of crosslinker was recently employed to


cause the transition of liposomes to inverted micelles. Liposomes, build from amphiphilic molecules with acid-labile
vinylether linkers, were shedding their PEG layer in acidic
conditions resulting in an accelerated drug release.81
pH-responsive swelling of particles
The total loss of micellar integrity is only one way to trigger the
drug release. The introduction of acid-sensitive groups into
nanoparticles can lead to swelling without complete disintegration of the structure. A key design feature of these nanoparticles
is the hydrophobic to hydrophilic transformation upon exposure to a mildly acidic environment with subsequent swelling.
This phenomenon can be observed upon increasing the hydrophilicity of one part of the copolymer. This swelling usually
allows the triggered release of the nanoparticles payload
by enabling faster diusion (Table 3). Chen et al. exploited
chets cyclic acetal to design pH-responsive biodegradable
Fre
micelles by copolymerizing cyclic aliphatic carbonate monomers with PEG-OH using ROP (Fig. 8, 26).82 The hydrolysis of
the acetal turned the hydrophobic polycarbonate into a more
hydrophilic structure, resulting in significant swelling of the
micelle. Fine-tuning the length of the PEG hydrophilic block
of the same copolymer system also led to the formation of
polymersomes.83 In both cases, the micellar structures were
maintained even after complete acetal hydrolysis, which was
assigned to the fact that the hydrophilic polycarbonate is not
fully water soluble. Nonetheless, the systems were successful
in releasing the drugs rapidly in response to mildly acidic
pH (Table 3). A similar behaviour was observed by Lu et al.

Composition and release eciency of swelling micelles and particles

Micelle size (nm)


System

Composition

Acid-sensitive group

pH 5

pH 7.4

Drug

Drug
loading (wt%)

26

Micelles

PEGpolycarbonate

1000

150

2683

Polymersomes

PEGpolycarbonate

1000

120

2784
2885

Micelles
cd-Particles

PDM-co-HEA
Homopolymer

Trimethoxybenzylidene
acetal
Trimethoxybenzylidene
acetal
Cyclic acetal
Trimethoxybenzylidene
acetal

800
900

167
100

PTX
DOX
PTX
DOX
Nile red
PTX

3/13
3/12
3/7
2/4
n.a.
1

Ref.
82

Releasea (%)
Time (h)

pH 5

pH 7.4

48

68
89
56
56
68
90

42
44
31
37
10
1

48
6
24

Values estimated from in vitro release profiles at both pH.

Fig. 8

2090

Structures of copolymers forming swelling particles at acidic pH.

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Their amphiphilic pH-sensitive copolymer poly(PDM-co-HEA)
(Fig. 8, 27) self-assembles into nanoparticles, which were able to
increase in size from nano-scale to almost micro-scale in acidic
environment due to the hydrolysis of the acid-labile acetals.84 Using
a dierent approach, Griset et al. reported the synthesis of expansible nanoparticles, which were prepared using a miniemulsion
polymerization technique. This approach combines high-energy
emulsification and free radical photopolymerization of a trimethoxybenzilidene acrylate monomer.85 The particles based on polymer 28
(Fig. 8) were cross-linked to be able to transform into a hydrophilic
nanogel structure at pH 5. The release of paclitaxel from the loaded
expansible nanoparticle was found to be pH dependent and related
to the hydrophobic to hydrophilic transformation.
Switching the solubility of dextrane particles
An acid-responsive biodegradable material was developed by
chet and coworkers based on acetal-derivatized dextran.86,87
Fre
Nanoparticles obtained from this material can be used as
triggered drug delivery systems thanks to its solubility switching
mechanism. Indeed, masking the hydroxyl groups of dextran as
acetals not only provides a hydrophobic material that can be
easily processed using various emulsion techniques, but also
provides a mechanism for introducing pH-sensitivity. Under
mildly acidic aqueous conditions, the pendant acetal groups
can hydrolyze, allowing a complete dissolution of the dextran
nanoparticles and their subsequent degradation (Fig. 9). Full
dissolution of the particles was observed after 24 h at pH 5,
whereas no degradation was observed at pH 7. This pH-dependent
degradation of Ac-DEX particles is further reflected in the
release profile of a model payload. Further studies on this system
demonstrated its eciency for the delivery of plasmid-DNA

ChemComm
and Si-RNA.88,89 More recently, the synthesis of ethoxy acetal
derivatized acetalated dextran has been reported, which presents the advantage of degrading into non-toxic products such
as ethanol, acetone, and dextran.90 Like Ac-DEX, Ace-DEX
microparticles have tunable degradation rates at pH 5 (intracellular) that can range from hours to several days by varying
the reaction time. These polymers could not only be used
to deliver traditional anti-cancer drugs but also hydrophobic
silver carbene complexes, which have strong anti-bacterial
properties.91

Polymerdrug conjugates
Besides encapsulation, another way to improve drug delivery is
by covalently attaching the drug to a macromolecular carrier.
Indeed, high molecular weight molecules and nano-sized
particles accumulate in solid tumours at much higher concentrations than in normal tissues or organs due to the Enhanced
Permeation and Retention (EPR) eect. Obviously, the stability
of the drugpolymer linkage is crucial. Based on the stability of
this bond, polymer conjugates may be classified into two broad
categories: permanent conjugates and prodrugs that usually
require the transformation of the prodrug to the drug within
the body to allow therapeutic action. Hence, the system should
be able to release the drug at the target site by using appropriate stimuli-responsive linkers. The literature on the release
of the drug from polymeric prodrugs by cleaving various
chemical linkages (esters, carbonates, carbamates, CN linkage,
and amides) has already been reviewed.92 Systems containing
acid-labile linkers, able to release the drug at slightly acidic pH,
have been quite popular for the design of intelligent carriers of
various architectures. Out of all acid-sensitive bonds, the hydrazone bond formed between the C13 carbonyl group of anthracyclines (i.e. Doxorubicin (Dox), daunomycin (Dau)) and polymer
hydrazides or the amide bond of a cis-aconityl residue containing
spacer is the most commonly used type for the preparation of
polymerdrug conjugates.
Linear polymerprotein conjugates

Fig. 9

Structure of dextran and its acetal-derivatives.

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The concept of macromolecular prodrugs, such as water


soluble polymerdrug conjugates, has been introduced by
Ringsdorf 30 years ago,93 and has been studied by many groups
since then. The most popular acid-sensitive system in that
category is probably the poly(N-(2-hydroxypropyl) methacrylamide)
(PHPMA)Doxorubicin (DOX) conjugate, formed through a
hydrolytically cleavable hydrazone bond with the keto group
of DOX in the C-13 position. Such graft polymer systems have
been extensively studied by Ulbrich and co-workers,8,94 and
have been proven to be ecient for therapeutic applications such
as chemotherapy and radiotherapy.95 The first example of HPMA
copolymers containing doxorubicin bound via pH-sensitive
linkage has been reported by Etrych et al. in 2001.96 While
the hydrazone bond is relatively stable at neutral pH, corresponding to the environment of the blood, the drug is released
under mildly acid conditions, such as in endosomes of tumour
cells, with half-lives in the order of hours. Based on this
approach, various PHPMA conjugates diering in the length
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Fig. 10

pHPMA hydrazide copolymer and keto-drugs commonly used for conjugation.

and structure of their spacer were studied, highlighting the


subtle relationship between the spacer structure and the rate of
DOX release.97 In vitro and in vivo studies on such systems
confirmed that this novel class of anticancer therapeutics
exhibits better pharmacokinetic and pharmacodynamic profiles and less side-toxicity in comparison with low molecular
weight drugs. More importantly, the polymeric conjugates
trigger an onset of specific anti-tumour immune response
and a profound therapeutic eect.98100 The HPMA grafted
copolymers have been more recently used in combination with
other therapeutics such as the anti-inflammatory and antiproliferative drug dexamethasone (DEX),101,102 as well as
the anticancer drugs paclitaxel (PTX), docetaxel (DTX),103 and
3-(9-acridinylamino)-5-hydroxymethylaniline (AHMA).104 In order
to obtain a hydrazone linkage, a ketone functionality needs to
be introduced on those drugs by esterification with levulinic
acid (LEV) or 4-(2-oxopropyl)benzoic acid (OPB) (Fig. 10), or by
selective acylation with thiazolidine-2-thione-activated carboxylic
acids in the case of AHMA. Table 4 sums up some drug release
experiments carried out by Ulbrich and co-workers for various
PHPMAdrug conjugates. Some studies still focus on the improvement of the PHPMADOX system by adjusting the molecular
weight, for instance by designing a high molecular weight graft
copolymer for enhanced passive tumour targeting,105 or by studying
the eect of narrow molecular weight distributions and
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Chem. Commun., 2013, 49, 2082--2102

well-defined molecular structures using RAFT polymerisation.106


Also, a thiol coupling strategy has been investigated for coupling
unmodified proteins/peptides lacking aldehyde/ketone functionality, or other thiol-containing molecules, to hydrazidederivatized PHPMA using heterobifunctional cross-linkers.107
Other candidates for the design of polymeric doxorubicin conjugates coupled via hydrazone functionality include poly(ethylene
oxide)108,109 and poly(methacryloyloxyethyl phosphorylcholine)
(polyMPC)110 as the main polymer.
Self-assembled systems
Although a substantial body of work is based on linear
copolymers, polymeric prodrugs can also be based on selfassembled micellar systems composed of amphiphilic block
copolymers. Indeed, in order to use polymeric micelles as drug
carriers, the loaded drug must be hydrophobic enough to
partition into the hydrophobic core of polymeric micelles.
The moderately water-soluble nature of some anticancer drugs
such as doxorubicin makes it dicult to physically load the
drug within the micelles in a sucient amount. This obstacle
can be overcome by conjugating the drug to the terminal end of
a hydrophobic polymer block. Acid-cleavable linkages, such as
hydrazone and cis-aconityl bonds, were first used for the
chemical conjugation of doxorubicin on a diblock copolymer of
polyethylene glycol-b-poly(L-lactic acid) by Yoo et al. (Fig. 11a).111
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Table 4

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Examples of drug release from various PHPMAdrug conjugates

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Releasea (%)
Ref.

Linker

Spacer (X)

Drug

Drug loading (wt%)

Mn PHPMA (g mol 1)

Time (h)

pH 5

pH 7.4

96

Hydrazone

DOX

Hydrazone
Hydrazone

97

Hydrazone

102
101

Hydrazone
Hydrazone

Gly
Gly-Gly
Gly-Phe-Leu-Gly
b-Ala
Aminobenzoic acid
Aminohexanoic acid
Aminohexanoic acid
Aminohexanoic acid

99
104

cis-Aconityl
Hydrazone

Gly-Phe-Leu-Gly
(Oxohexyl)methacrylamide

9.5
10.5
10.2
7.6
8.2
7.9
9.5
10.5
6.8
6.8
4.7
2.3
5
6.6
4.4
6.3

25 700
19 600
38 900
33 500
34 200
15 800
25 700
21 100
25 600
38 400
35 700
30 000
41 000
56 000
38 800
21 500

48

106
103

Gly-Gly
Gly-Phe-Leu-Gly
Aminohexanoic acid
Aminohexanoic acid

85
75
80
90
80
85
85
75
71
92
96
93
90
86
75
98

10
5
3
38
5
11
10
6
7
16
5
22
42
50
3
8

DOX
PTXLEV
DTXLEV
DOX

DEXOPB
DEXLEV
DEXOPB
DOX
AHMA

9
24
48

6
24
48
24

Values estimated from in vitro release profiles at both pH.

The doxorubicin conjugated micelles were about 89 nm in diameter


and their critical micelle concentration was 1.3 mg ml 1.
Moreover, doxorubicin micelles showed more cytotoxicity
against cancer cells than free doxorubicin. This strategy has
also been used and thoroughly studied by Kataoka and co-workers
with the design of pH-sensitive polymeric micelles based on

Fig. 11

poly(ethylene glycol)poly(aspartate hydrazide) [PEGp(Asp-Hyd)]


block copolymers, synthesized by ring-opening polymerization of
b-benzyl-L-aspartate N-carboxy-anhydride (BLANCA) from an
amino-terminated linear PEG chain (Fig. 11b).112 Indeed, Bae
and co-workers applied this system to the delivery of various
anticancer drugs (Table 5).113118 Influence of various parameters

Structure of block-copolymerdrug conjugates prior to micellar self-assembly.

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Table 5

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Composition and release eciency of PEGP(Asp)-based micelles (Fig. 11b)

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Releasea (%)
Ref.

Spacer

PEGPAsp

Drug

Drug loading (wt%)

Micelle diameter (nm)

Time (h)

pH 5

pH 7.4

113
114

1237
1238

67.6%/asp
>31 wt%

65
o200

24
24

115

1240

Gly

125
1215
1235
125
1215
1235
125
1215
1235
1235
533
1235

42
137
85
44
45
40
11
25
43
86
62
44
38
38
259

24

116

ADR
DOX
GDMLEV
PTXLEV
PTXOPB
1 : 1 co-assembly
DOX

25
60
81
57.6
0
44.9
40
57
37
60
65
o30
78
45
45
45
55
58

o5
20
78
29.2
0
34
30
44
17
36
30
o30
39
10
38
28
40
49

Abz
117

118
a

Ester only
4-Acetylbutyric acid
6-Oxoheptanoic acid
7-Oxooctanoic acid

DEX

3PO

37.50%
87.50%
13
2.8
10.4
31.6
4.1
10.9
11.4
8.7
4.6
5.6
4.5
2.08
2.21

48

24

Values estimated from in vitro release profiles at both pH.

such as the poly(aspartate) block length and the nature of the linker
on the micelles size and release profile was investigated. It appears
that the drug loading was highly depending on the nature of the
drug and the linker. Moreover, the release time of those micellar
systems was overall slower than for the graft copolymers systems
(Table 5), and in general the dierences in release rate between
pH 5.5 and 7.4 was less noticeable. This can be explained by the
presence of the hydrophilic shell that protects the hydrazone linker
from the external media. Particularly, the micellar system designed
for the delivery of the glycolytic enzyme inhibitor, 3-(3-pyridinyl)-1(4-pyridinyl)-2-propen-1-one (3PO), showed only a small dierence
in the release profile at both pH after 3 hours, with a significant
drug release at pH 7.4. These results indicate that the micelles may
retain 3PO in the blood stream (pH 7.4) only for a short time. It is
also interesting to note that the method used for dialysis was found
to have a large influence on the drug release profiles.112 In vitro and
in vivo studies performed on the adriamycin-loaded system show
the eciency of this micellar system including the intracellular
pH-triggered drug release capability, tumour-infiltrating permeability as well as eective antitumor activity with extremely low
toxicity.119 A step further in the design of pH-sensitive polymeric
micelles as drug carriers is the introduction of targeting agents by
modification of the polymer backbone with piloting molecules for
cancer cells targeting. Bae et al. paved the way to such systems by
introducing folate (Fol) at the end of the shell-forming PEG chain to
enhance intracellular transport.120 More recently, Xiong et al. conjugated doxorubicin to poly(ethylene oxide)-block-poly(3-caprolactone)
(PEO-b-PCL) micelles decorated with the avb3 integrin targeting
ligand RGD4C on the micellar surface (Fig. 11c).121 The presence of
the targeting ligand allowed the accumulation of DOX in the
nucleus of sensitive cells and mitochondria of resistant cells.
Moreover, the micellar systems were found to be more eective
than free DOX in vivo in inhibiting the growth of DOX sensitive and
resistant tumours, respectively. The same team also proposed an
improved version of this system by designing a virus mimetic shell
that was conferred by attaching two ligands, i.e., the integrin
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Chem. Commun., 2013, 49, 2082--2102

specific ligand RGD4C for active cancer targeting and the


cell-penetrating peptide TAT for membrane activity, to eciently
co-deliver siRNA and DOX.122 Multifunctional folate decorated
micelles were also obtained by Hu et al. by co-assembling a PEGb-PLADox conjugated copolymer and a rhodamine B-conjugated
copolymer (PEG-b-P(LA-co-ME/RhB)) together with folate terminated block copolymer (FAPEG-b-PLA) (Fig. 11d).123 In parallel to
PHPMA based statistical copolymers, Ulbrich and colleagues also
designed an acid-degradable micellar system for the delivery of
DOX, using poly(ethylene oxide)-block-poly(allyl glycidyl ether)
(PEOPAGE) copolymers (Fig. 11e)124,125 and cholesterol-based
PHPMA copolymers.126 Recently, Binauld et al. reported the synthesis of micellar acid-degradable polymerplatinum conjugates by
postmodification of a POEGMEMA-b-PHEMA block copolymer
obtained by RAFT polymerization (Fig. 11f).127 Other examples of
self-assembled hydrazone-based acid-degradable prodrugs include
small micelles from amphiphilic macromolecules,128 and vesicles
formed by heterofunctional triblock copolymer for combined
tumour-targeted delivery of an anticancer drug and superparamagnetic iron oxide nanoparticles.129
Cross-linked micellar prodrugs
Recent work on polymeric micelles systems has been focused
on stabilization strategies to overcome premature dissociation
of micellar drug carriers in in vivo conditions. In the case of
micellar polymerdrug conjugates, some strategies have been
developed to introduce a second trigger in the system or to use
a drug derivative as a cross-linking agent. For instance, Wei
et al. used the free hydrazide group to crosslink the ADRconjugated system using dithiodiethanoic acid as a cross-linker
(Fig. 12a).130 Such cross-linked micelles take advantage of two
intracellular chemical triggers, namely, acid and disulfide
reduction, for the controlled release of ADR. It should exhibit
an eective release only when both low pH and disulfide
reduction conditions are present. Cross-linking via disulphide
bond was also achieved by Jia et al. using a dierent approach.131
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Table 6 Composition and release eciency of cross-linked micellar prodrugs
(structures from Fig. 12)

Ref. Polymer

Drug

130 PEO-b-PMAA ADR


131 P(PDSM)DOX
b-P(HPMA)
132 PEG-bDOX
P(HPMA-Lac)
133 PEG-b-PLA
Cisplatin

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Fig. 12 Structures of the polymers and cross-linkers precursors of the crosslinked micellar prodrugs.

In a one-pot reaction, the versatility of PDS groups on amphiphilic PPDSM-b-PHPMA, synthesized by RAFT polymerization,
was used to conjugate doxorubicin and simultaneously crosslink
the micellar assemblies via acid-cleavable hydrazone bonds and
reducible disulfide bonds (Fig. 12b), allowing the system to be
cleaved into unimers in a reductive intracellular environment.
Core-cross-linked biodegradable polymeric micelles composed of
poly(ethyleneglycol)-b-poly[N-(2-hydroxypropyl) methacrylamidelactate] (mPEG-b-P(HPMAmLacn)) were obtained by Talelli
et al. by polymerizing a DOX methacrylamide derivative using
free radical polymerization (Fig. 12c).132 The entire drug payload
was released within 24 h incubation at pH 5 whereas only around
5% release was observed at pH 7.4 (Table 6). Finally, Aryal et al.
proposed an original system for the controlled delivery of cisplatin,
by designing novel acid-responsive Bi(PEGPLA)Pt(IV) polymer
cisplatin prodrug conjugate nanoparticles (Fig. 12d).133 The
resulting particles showed excellent acid-responsive drug release
characteristics and potent cytotoxicity against ovarian cancer.
Polysaccharides
Due to their excellent physicochemical properties, biocompatibility
and biodegradability, polysaccharides have also been investigated
for developing macromolecular prodrugs. More specifically, the
design of derivatives with tuneable bio-responsiveness, targeting
or environmental triggering properties, has raised interest in the
past few years. One of the first examples of chemical conjugation
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The Royal Society of Chemistry 2013

Drug
Micelles
Releasea (%)
loading diameter Time
pH 5 pH 7.4
(wt%) (nm)
(h)
11
24.9

56
61

24
24

25
72

14
21

3040

80

24

100

86

24

83

55

Values estimated from in vitro release profiles at both pH.

of an anticancer drug to a polysaccharide via an acid-degradable


bond was reported by AI-Shamkhani and Duncan, who synthesized conjugates of alginate and the antitumor agent daunomycin
(DNM).134 High (Mw = 250 000 g mol 1) and low (Mw =
61 000 g mol 1) molecular weight alginateDNM conjugates
were prepared, with a low drug loading. Administration of LMW
alginateDNM conjugate resulted in a delay in tumour growth
and reduced toxicity when compared to free DNM. More recently,
Scomparin et al. reported the design of new polymer therapeutics
based on bioconjugation of DOX on pullulan via a hydrazone
linker.135 The polymer bioconjugation was found to prolong the
circulation time of the drug in vivo. More recently, low molecular
weight hyaluronan (LMWHA) oligomers were introduced on the
HPMA copolymer for enhanced targeting properties.136 The use
of a cis-aconityl acid-degradable linker also seems to be popular
for the design of such systems. For instance, Hu et al. synthesized
DOX conjugated stearic acid-g-chitosan oligosaccharide polymeric micelles using the acid-degradable cis-aconityl bond.137
The elevation of the pH resulted in a slightly decreased liberation
of the drug from the conjugates, which was studied using various
drug contents (Table 7). Son et al. described the preparation of
glycol-chitosanDOX nanoaggregates containing cis-aconityl
spacers. The GCDOX formed spontaneously micelle-like nanoaggregates in aqueous media with a diameter of about 250 nm.138
The resulting particles exhibited anti-tumour activity via EPR
eect. A similar system was employed later for the delivery of
adriamycin. Interestingly, the release of the drug was faster
(Table 7).139 However, free adriamycin showed in that case more
potent cytotoxicity than the conjugates.
Other architectures
Besides linear statistical copolymers and self-assembled micelles,
other systems can be used as reactive polymer scaold for
anticancer drug conjugation via an acid-degradable linker.
Examples include dendrimers, hyperbranched and star polymers, gold nanoparticles and more. Table 8 summarizes some
recent examples for the controlled release of doxorubicin, in
which the core is functionalized for the chemical conjugation
of DOX via a hydrazone or cis-aconityl linker and the shell is a
biocompatible hydrophilic polymer. Calderon et al. prepared
dendritic polyglyceroldoxorubicin prodrugs, which were
further post-modified with a poly(ethylene glycol) shell.140
The resulting system displayed pronounced acid-sensitivity,
good cellular internalization and a favourable toxicity profile.
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Table 7

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Composition and release eciency of the glycopolymerdrug conjugates

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Releasea (%)
Ref.

Linker

System

Drug

134

Hydrazone

Alginate
LMW
HMW
Pullulan particles
Stearic
Acid-g-chitosan micelles

DNM

135
137

Hydrazone
cis-Aconityl

138
139

cis-Aconityl
cis-Aconityl

Glycol-chitosan nanoaggregates
Glycol-chitosan nanoaggregates

DOX
DOX
DOX
ADR

Drug loading (wt%)


0.8
1.3
6
3%
6%
10%
5
5

Particle diameter (nm)

Time (h)

n.a.

48

100150
40
70
106
238
238

48
48
48
48

pH 5

pH 7

63
23
100
42
37
28
9 (pH 4)
26 (pH 4)

3
3
10
31
28
15
2.5
6

Values estimated from in vitro release profiles at both pH.

Table 8

Composition and release eciency of hyperbranched micellar prodrugs and other nanoparticles

Releasea (%)
Ref.

Linker

Core

Shell

DOX loading (wt%)

Particle diameter (nm)

Time (h)

pH 5

pH 7

140
141
142 and 143
145 and 146

Hydrazone
Hydrazone
Hydrazone
cis-Aconityl

Dendritic polyglycerol
Dendritic polyglycerol
PAMAM dendron
PAMAM dendron

PEG
PEG
HPMA
PEG

Hydrazone
Hydrazone
Hydrazone
Hydrazone

Boltorn H40
Gold nanoparticle
Boltorn H40
PDA capsule

PEG-b-PLasp
PEG-b-PLasp
PEG-b-PLGlu
PMA

16.4
183
n.a.
83
18
16
50
2452
4491
300

24
24
24
48

147
150
148
149

5
2.1
11
1/4 18
1/16 8.5
1/32 6.4
16
17
16.2

75
70
90
4
8
24
76
70
85
85

o5
50
5
o1
o1
o1
12
10
10
20

24
24
24
12

Values estimated from in vitro release profiles at both pH.

A similar system was designed later on by Lee et al. starting


from a PEG-b-polyglycerol block copolymer.141 Ulbrich and
colleagues used their knowledge on the acid degradable
pHPMADOX conjugate to design equivalent star polymers with
the core formed by poly(amidoamine) (PAMAM) dendrimers
grafted with semitelechelic N-(2-hydroxypropyl)methacrylamide
(HPMA) copolymers.142,143 The polymer arms were attached to
the dendrimers through either stable amide bonds or via enzymatically or reductively degradable spacers, which enabled intracellular degradation of the high-molecular-weight polymer carrier.
Moreover, star polymer conjugates showed higher in vivo antitumour activities than the free drug or linear polymer conjugate
when tested in mice bearing EL4 T-cell lymphoma. These star
polymers were also compared to linear analogues to study their
in vivo distribution, elimination and tumour accumulation using
a fluorescent and cleavable model drug.144 The study showed the
superiority of the star polymers in terms of circulation time in the
body. PAMAM dendrons were associated to a PEG shell by Zhu
et al., in which DOX was conjugated through acid-sensitive cisaconityl linkage.145 Antitumor activity of the conjugates was
proved to increase along with the PEGylation degree. The release
rate of DOX appeared to be rather slow, even at acidic pH. The
same carrier was later on conjugated to RGD as a targeting agent,
which consequently improved the therapeutic eect against
murine B16 melanoma, compared to the previous system.146
Prabaharan et al. conjugated an amphiphilic block copolymer to
two types of core: commercial hyperbranched aliphatic polyester
(Boltorn H40),147 and gold nanoparticles.147 Both systems contained an hydrophobic poly(L-aspartate-doxorubicin) inner shell,
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and a hydrophilic and folate-conjugated poly(ethylene glycol)


outer shell. DOX was covalently conjugated onto the hydrophobic segments of the amphiphilic block copolymer chains by
pH-sensitive hydrazone linkage. In both cases, the release profiles
showed a strong dependency on the environmental pH values.
The same group also investigated a system based on H40-poly(L-glutamate-hydrazone-doxorubicin)-b-PEG that was conjugated
to RGD peptides and macrocyclic chelators to design multifunctional unimolecular micelles for cancer-targeted drug delivery
and PET imaging.148 An original approach was recently developed
by Carusos group, who immobilized DOXpoly(methacrylic acid)
(PMAA) conjugates onto mussel-inspired polydopamine (PDA)
nanocapsules via robust thiolcatechol reactions.149 Cell viability
assays demonstrated the enhanced eectiveness of DOX-loaded
PDA capsules in eradicating HeLa cancer cells, compared with
free Dox under the same assay conditions.

PolymerDNA complexes
Gene therapy holds great promise for the treatment of severe
diseases including cancer. In recent years the design of synthetic
carriers for nucleic acid delivery has become a research field of
increasing interest. Viral vectors that have been commonly used
are limited by their cargo loading and, more importantly, may
lead to immune reactions. In this context non-viral carriers for
plasmid DNA and Si-RNA have gained increasing interest in gene
therapy research during the past few years.151 The new trend is
now to design targeting and bio-degradable devices that will
allow improved eciency and lower side eects.
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Degradable PEI-based polyplexes


Among the cationic polymers, polyethylenimine (PEI), a commercially available cationic polyamine, is one of the most
successful and widely studied gene delivery polymers. However,
the design of vectors that mediate gene transfer eciently but do
not cause appreciable toxic side-eects is still a major challenge
in this field. Indeed, transfection eciency and cytotoxicity of
PEI depend on its molecular weight. For instance, PEI with a
higher molecular weight (i.e. 25 kDa) shows high transfection
eciency and cytotoxicity. On the other hand, PEI with a lower
molecular weight (i.e. 1.8 kDa) shows low transfection and
cytotoxicity.152 Therefore, optimization can be achieved of the
adequate sizes of the biodegradable polycations, which allow
gene transfer as eective as their stable PEI counterparts but can
decompose into nontoxic degradation products.
To that purpose, Kim et al. synthesized degradable PEIs with
acid-labile imine linkers from low molecular weight branched PEI
(1.8 kDa) and glutardialdehyde (Table 9).153 An in vitro transfection assay showed that the transfection eciency of the acid-labile
PEIs was comparable to that of PEI25K. The toxicity assay reveals
however that the acid-labile PEI was much less toxic than PEI25K,
due to the degradation of acid-labile linkage. Knorr et al. applied a
similar strategy combining acetal cross-linkers MK and BAA
(Table 9) with linear oligoethyleneimine (OEI).154 Polyplexes with
acid-labile polymers showed an improved toxicity profile compared to those made with acid-stable polymer analogues. At low
cation/plasmid (c/p) w/w ratios the transfection eciency of
pH-sensitive polymers was slightly reduced, but it became similar
or superior to the eciency of acid-stable polymers at higher c/p
ratios. The same group also used the MK cross-linker to copolymerize OEI with PEG, leading to dynamic PEGpolycation copolymers. The presence of PEG allows pH-responsive reversible
surface shielding of the polyplex.155 In order to improve Si-RNA
delivery from linear PEI, Shim and Kwon incorporated aciddegradable amino ketal branches to secondary amines of linear
PEI (Table 9) to facilitate endosomal escape and the subsequent
release of Si-RNA to the cytoplasm.156 The conjugation of aciddegradable amino ketal branches resulted in enhanced cytoplasmic release of free Si-RNA while preserving low cytotoxicity.
The same process was applied to branched PEI, showing enhanced
transfection and RNA interference as well as remarkably reduced
cytotoxicity.157 Benefits of ketalized polymers as stimuli-responsive
Si-RNA carriers are highlighted in a recent review of Kwon.158
Dynamic PEG-shielding
Conjugation of poly(ethylene glycol) (PEG) to the polycations
has been proven to be an eective way to prolong the circulation
Table 9

duration of the cationic delivery systems, e.g., polycationgene


complex, by shielding the surface charge of the polycation
moiety. However, this approach also diminishes the cellular
uptake. Connecting PEG chains via an acid cleavable linker
would allow the detachment of PEG in endosomes in order
to reserve the intracellular delivery eciency. Meanwhile,
shielding of the positive charges prolongs the circulation
duration that enables the carrier to target remote sites. Therefore, dynamic PEGpolycation copolymers that release PEG and
degrade into small fragments after entering the cell were
developed by Knorr et al. using ketalized PEIPEG copolymers
(Fig. 13a).155 Compared to the non-PEGylated polyplex, the
acid-labile PEGylation eciently reduced toxicity, but interestingly also improved transfection eciency of the OEI-based
polymer. Murthy et al. reported the synthesis of graft terpolymers that consist of a hydrophobic, membrane-disruptive
backbone onto which hydrophilic PEG chains have been
grafted through acid-degradable acetal linkers (Fig. 13b).159
These so-called encrypted polymers eciently delivered oligonucleotides and macromolecules into the cytoplasm of hepatocytes. An orthoester pH-sensitive linker between the PEG and
the hydrophobic chain of PEG lipids was used by Masson et al.
for the design of stabilized lipoplex (Fig. 13c).160 Once again,
in vitro results showed a significant increase in transfection
with formulations containing pH-sensitive PEG lipids versus
non-pH-sensitive analogues. Gu et al. introduced a PEG shielding
moiety on cationic micelles via the intermediate of a novel pH
responsible benzoic imine linker (Fig. 13d).161 The micelles
showed better biocompatibility at physiological pH while they
became membrane disruptive at a pH lower than the extracellular
pH of the tumour. More examples of dynamic PEGpolycation
conjugates can be found in the review of E. Wagner.152
Advanced DNA/Si-RNA carriers
Similar to drug delivery carriers for low molecular weight drugs,
smart nanovectors for DNA delivery can take on various forms,
ranging from grafted cationic polymers to self-assembled systems and nanocapsules. A significant amount of research has
focused in these past two years on the design of systems with
improved degradation, transfection and targeting properties.
Besides PEI based-polyplexes, Kwon and Shim developed new
grafted cationic polymers bearing an acetal linker on the sidechain to trigger the release of the DNA when entering the cell.
A poly(amino ester) with a ketal functionality was synthesized,
that presents the ability to degrade via rapid hydrolysis of
the ketal branches in the mildly acidic endosome, but the polyester backbone can also subsequently gradually degrade.162

Degradation of PEI polyplexes

Ref.

Acid-degradable linker

System

Polymer

pH

Polymer half-life (h)

153
154

Imine
Acetal

Polyplexes with DNA


Polyplexes with DNA

4.5/7.4
5/7.4

155
156

Acetal
Acetal

Polyplexes with DNA


Polyplexes with SiRNA

157

Acetal

Polyplexes with DNA or SiRNA

Branched PEI
OEIMK
OEIBAA
PEGOEI copolymer
Linear PEI LMW
Linear PEI HMW
Branched PEI

1.1/118
0.25/5
3 min/3.5
n.a.
2.3/21.6
1.9/20.3
2.6/26.4

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Fig. 13

Structures of polymers allowing dynamic PEG-shielding.

Fig. 14

Examples of acid-degradable polymers used for pDNA triggered release.

Also, a monomer-to-polymer approach was used to obtain aciddegradable cationic nanoparticles,163 using an aliphatic monomer with a pendant primary amine group and a cleavable acetal
chets group
linkage, which was developed previously in Fre
(Fig. 14a).48 This approach allows highly flexible nanoparticle
fabrication, resulting in higher transfection eciency by the
degradable nanoparticles than PEI polyplexes at very low concentrations. Micelles that can undergo structural transformations at
low pH value were obtained by Shim and Kwon by self-assembling
DNA with PEG-conjugated poly(ketalized serine) (Fig. 14b).164
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Upon acid-catalyzed hydrolysis of the ketal linkages, poly(kSer) converts to neutral and naturally occurring poly(serine),
resulting in the destabilization of the micelles. In addition, the
study employed core cross-linking via acid-cleavable aminebearing branches to improve the transfection capability.
A dierent approach was proposed by Chen et al. to promote
the transfection eciency of p-DNA by encapsulating the
PEIDNA polyplexes into microparticles of biodegradable
polymers containing acid-labile segments and galactose grafts
(Fig. 14c).165 The presence of galactose moieties also enhanced
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Conclusions

Fig. 15 Degradable, pH-sensitive, membrane-destabilizing, comb-like polymers


for nucleic acids delivery.166

cellular uptake of microspheres and increased acid-lability due


to the increased hydrophilicity of the acid-labile segments in
the presence of the sugar.
A good example of a smart advanced carrier for Si-RNA
delivery was designed by Lin et al. (Fig. 15).166 Based on selfassembled comb-like polymers, the block copolymer incorporates a first block based on pH-sensitive ethylacrylic acid (EAA)
monomers and hydrophobic hexyl methacrylate monomers
(HMA) with membrane-destabilizing activity and a second block
bearing, via an acid-cleavable hydrazone linker, grafts of hydrophobic HMA and cationic trimethylamino-ethylmethacrylate
(TMAEMA) monomers, which can condense DNA/RNA. The
membrane-destabilizing backbone and the hydrophobic
monomers embedded in the comb-like grafts should synergistically disrupt the endosomal membrane and release the
nucleic acid cargo into the cytoplasm to produce the desired
therapeutic activity. Moreover, the macromolecule can degrade
into smaller fragments in an acidic environment, which will
minimize their toxicity. Another interesting approach was
developed by Kulkarni et al., who used a hostguest-derived
delivery vector based on the self-assembly of cationic b-cyclodextrin
derivatives with a poly(vinyl alcohol) main chain polymer bearing
PEG. Adamantane or cholesterol was conjugated to the polymer
using an acid-sensitive benzylidene-acetal functional group
(Fig. 16).167,168 These systems were investigated for their ability
to promote supramolecular complex formation with pDNA- and
Si-RNA-cyclodextrin derivatives. In vitro studies showed that
they had 103-fold lower cytotoxicities than 25 kDa bPEI, while
maintaining higher transfection eciencies.

Fig. 16

This review provides a large overview of the recent acid-degradable


polymeric systems, from the macroscale to the nano/microscale.
As it appears, each kind of linker finds its application in a given
field, depending on the synthetic possibility allowed by each of
them. Indeed, acetal and orthoesters are linkers of choice for the
design of linear degradable polymers, whereas the hydrazone
bond is usually chosen to obtain hydrogels with tuneable properties. Acetals are also advantageous for the design of ecient aciddegradable cross-linkers, as well as smart nanoparticles and
nanogels. On the other hand, hydrazone and cis-aconityl coupling
still lead the way to obtain acid-cleavable polymerdrug conjugates of various architectures. The last decade has paved the way
for a new generation of intelligent drug carriers, with tailored
structures and delivery properties. A wide selection of micro or
nano-devices are using the natural pH changes occurring in the
body to trigger the drug release, either by degradation disintegration of the carrier or cleavage of the polymerdrug bonding. Some
chet and coworkers on
pioneering work has been done by Fre
acetal-based systems for anticancer drug delivery, by Kwon and
colleagues for ketalized polymerDNA polyplexes, and by Ulbrich
and Baes groups on hydrazone-based systems. From those solid
bases are emerging exciting new architectures, allowing the
delivery of an increasing number of therapeutics including anticancer drugs, DNA and SiRNA. A further step has now been taken
to excel beyond simple drug delivery devices towards theranostics
approaches, that combine therapy and imaging in one platform.
Features such as MRI contrast agents169 incorporated into acid
degradable polymers present the next generation materials.
Giving the exponential interest for such acid-sensitive systems, the risk is now to decline the same strategies indefinitely
without going to the next step that would be the clinical trial.
A recent review by Duncan and Richardson170 highlighted the
advancements and challenges of nanomedicine. Chemists have
now provided a full toolbox to create materials in dierent
shapes and sizes, which can degrade or change properties at
preset times. Further developments rely on close collaborations
between dierent disciplines. A range of aspects need to be
considered to create a material that may be competitive on the
market. While its properties should present a significant advancement as drug delivery platform compared to already available
polymers, other aspects such as safety and cost-eectiveness can
decide on the fate of the technology. Some of the degradation

Mechanism of compaction of nucleic acid AdPVAPEG cargo into stable nanometer-size particles and its degradation.167

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products listed in this review may fail this bill while other
approaches may be too expensive or dicult to upscale. However, this should not discourage the reader to reach into this
toolbox and use this remarkable chemistry. The opportunities
are evident and highly promising.

Acknowledgements

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Published on 10 December 2012 on http://pubs.rsc.org | doi:10.1039/C2CC36589H

MS thanks the ARC (Australian Research Council) for funding


in the form of a Future Fellowship (FT0991273) and a Discovery
Project (DP1092694).

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