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EXPERT REVIEW
Click Chemistry with Polymers, Dendrimers, and Hydrogels
for Drug Delivery
Enrique Lallana &Francisco Fernandez-Trillo &Ana Sousa-Herves &Ricardo Riguera &Eduardo Fernandez-Megia
Received: 16 November 2011 / Accepted: 6 January 2012 / Published online: 25 January 2012
#Springer Science+Business Media, LLC 2012
ABSTRACT During the last decades, great efforts have been
devoted to design polymers for reducing the toxicity, increasing
the absorption, and improving the release profile of drugs.
Advantage has been also taken from the inherent multivalency
of polymers and dendrimers for the incorporation of diverse
functional molecules of interest in targeting and diagnosis. In
addition, polymeric hydrogels with the ability to encapsulate drugs
and cells have been developed for drug delivery and tissue
engineering applications. In the long road to this successful story,
pharmaceutical sciences have been accompanied by parallel
advances in synthetic methodologies allowing the preparation of
precise polymeric materials with enhanced properties. In this
context, the introduction of the click concept by Sharpless and
coworkers in 2001 focusing the attention on modularity and
orthogonality has greatly benefited polymer synthesis, an area
where reaction efficiency and product purity are significantly chal-
lenged. The purpose of this Expert Review is to discuss the impact
of click chemistry in the preparation and functionalization of
polymers, dendrimers, and hydrogels of interest in drug delivery.
KEY WORDS click chemistry.dendrimer.drug delivery.
hydrogel .polymer
ABBREVIATIONS
AIBN azobisisobutyronitrile
ATRP atom transfer radical polymerization
bis-MPA 2,2-bis(hydroxymethyl)propionic acid
BPDS bathophenanthroline disulphonated disodium salt
CA contrast agent
CL caprolactone
ConA Concanavalin A
CPT camptothecin
CuAAC Cu(I)-catalyzed azide-alkyne cycloaddition
DBU 1,8-diazabicyclo[5.4.0]undec-7-ene
DDS drug delivery system
DIPEA N,N-diisopropylethylamine
DMPA 2,2-dimethoxy-2-phenylacetophenone
DOX doxorubicin
EPR enhanced permeability and retention
GATG gallic acid-triethylene glycol
LCST lower critical solution temperature
LRP living radical polymerization
MAPC methacryloyloxyethyl phosphorylcholine
MMP matrix metalloproteinase
MRI magnetic resonance imaging
MSC mesenchymal stem cells
NMP N-methyl-2-pyrrolidone
PAMAM poly(amido amine)
PEG poly(ethylene glycol)
PEI poly(ethylene imine)
PEO poly(ethylene oxide)
PIC polyion complex
PLL poly-L-lysine
PMA propargyl methacrylate
PMDETA N,N,N′,N′,N″-pentamethyldiethylenetriamine
PMMA poly(methyl methacrylate)
PNIPAM poly(N-isopropylacrylamide)
POEGA poly(oligo(ethylene glycol) acrylate)
PPI poly(propylene imine)
PS poly(styrene)
PVA poly(vinyl alcohol)
E. Lallana :F. Fernandez-Trillo :A. Sousa-Herves :R. Riguera :
E. Fernandez-Megia (*)
Department of Organic Chemistry
Center for Research in Biological Chemistry &
Molecular Materials (CIQUS)
University of Santiago de Compostela
Jenaro de la Fuente s/n
15782, Santiago de Compostela, Spain
e-mail: ef.megia@usc.es
Pharm Res (2012) 29:902–921
DOI 10.1007/s11095-012-0683-y
RAFT reversible addition-fragmentation chain transfer
RGD Arg-Gly-Asp
ROMP ring-opening methathesis polymerization
ROS reactive oxygen species
SPAAC strain-promoted azide-alkyne cycloaddition
SPR surface plasmon resonance
TBTA tris(benzyltriazolylmethyl)amine
TEC thiol-ene coupling
THPTA tris(hydroxypropyltriazolylmethyl)amine
TMS trimethylsilyl
TYC thiol-yne coupling
The reaction must be modular wide in scope, give very high yields,
generate only inoffensive byproducts that can be removed by non-
chromatographic methods, and be stereospecific (but not necessarily
enantioselective). The required process characteristics include simple
reaction conditions (ideally, the process should be insensitive to oxygen
and water), readily available starting materials and reagents, the use
of no solvent or a solvent that is benign (such as water) or easily
removed, and simple product isolation. Purification - if required -
must be by nonchromatographic methods, such as crystallization or
distillation, and the product must be stable under physiological
conditions…Click processes proceed rapidly to completion and also
tend to be highly selective for a single product: we think of these
reactions as being “spring-loaded”for a single trajectory”.H.C.
Kolb, M. G. Finn and K. B. Sharpless. Angew. Chem., Int. Ed. 40:
2004–2021 (2001).
INTRODUCTION
Since the mid-1960s, great efforts have been devoted to
the development of drug delivery systems (DDS) for the
controlled administration of drugs (1). Encouraged by
breakthroughs in the field [PEGylation, active targeting,
enhanced permeability and retention (EPR) effect] and
the need of more sophisticated materials and novel
designs, chemists have perceived drug delivery as an
attractive field to collaborate with colleagues in the
pharmaceutical and medical sciences. Indeed, the ability
of putting together small building blocks into larger
structures has been at the core of evolution and inspired
chemists in the search of more efficient processes with
production of minimal waste (2). In this context, Sharp-
less and coworkers introduced in 2001 the concept of
click chemistry in an effort to focus the attention on the
easy production of properties rather than on challenging
structures (3). The idea behind click chemistry is to
deliver new avenues for the preparation of useful mate-
rials from readily available building blocks and extreme-
ly efficient chemical transformations. During the last
decade the click philosophy has received a warm wel-
come by researchers in different fields and inspired the
publication of hundreds of papers in areas such as,
materials and polymer science, nanotechnology, and
drug delivery and the pharmaceutical sciences in gener-
al (4–7). In a previous Expert Review, we have critically
discussed the click concept, its advantages, uses and
misuses, as well as its application for the preparation
and functionalization of nanosized DDS (8). As the vast
majority of these examples rely on the Cu(I)-catalyzed
azide-alkyne cycloaddition (CuAAC) (9,10), a section
dealing with the use of this reaction in bioconjugation
was provided, covering the role of Cu in the structural
damage of biomolecules, the use of Cu(I)-chelating
ligands (Fig. 1), and the development of the Cu-free
strain-promoted azide-alkyne cycloaddition (SPAAC)
(11). More recently, a detailed perspective on the effi-
cient application of CuAAC and SPAAC for the in vitro
functionalization of biomacromolecules has been pub-
lished by our group (12).
During the last decades, a great deal of work has been
devoted to adapt, modify, or tailor polymers for reducing
drug toxicity, increasing drug absorption, and improving
drug release profiles (13). These efforts have indeed benefit-
ed from the emergence of the click concept. Thus, although
the main application of click chemistry originally envisaged
by Sharpless and coworkers was oriented to drug discovery,
the click concept has profoundly impacted polymer synthe-
sis, an area where reaction efficiency and product purity are
significantly challenged (14). In this Expert Review a survey
Fig. 1 Cu(I)-chelating ligands for CuAAC commonly employed in bio-
conjugation. [BPDS (bathophenanthroline disulphonated disodium salt);
TBTA [tris(benzyltriazolylmethyl)amine]; THPTA [tris(hydroxypropyltriazo-
lylmethyl)amine]].
Clicking Polymers for Drug Delivery 903
of the most recent applications of CuAAC and other click
reactions, including Michael addition, Diels-Alder, thiol-ene
coupling (TEC), and SPAAC for the preparation and func-
tionalization of dendrimers, synthetic polymers, and hydro-
gels will be discussed with special emphasis on those
examples of relevance in drug delivery.
DENDRIMERS
Dendrimers are perfectly monodisperse macromolecules
made of branched repeating units emerging from a central
core, that are characterized by a high degree of functionality
(15). In general, they have globular shape with diameters in
the nanometer scale (ca. 1–20 nm), which makes them ideal
candidates for bio- and nanotechnology applications (16).
The branched architecture of dendrimers demands rigorous
synthetic protocols for their preparation, which normally
involve activation/growing steps and tedious purifications.
In this context, it is not surprising that the preparations of
some of the most successful dendritic scaffolds have relied on
click reactions appeared before the click concept being
proposed. This is for example the case of poly(amido amine)
(PAMAM) (17) and poly(propylene imine) (PPI) (18) den-
drimers, which rely on Michael additions in their growing
steps, or glycerol dendrimers which use a dihydroxylation
reaction as activation step (19). In this section we will focus
the attention on reports that adopting the original paper by
Sharpless (3) as source of inspiration have made significant
contributions to the preparation and functionalization of
dendrimers for drug delivery applications.
Synthesis of Dendrimers
The first application of CuAAC to the synthesis of den-
drimers was reported in 2004 as a result of collaboration
between the groups of Hawker, Voit, Fréchet, Sharpless,
and Fokin (20). A convergent CuAAC-based approach was
employed for the preparation of triazol dendrimers mimick-
ing the earlier synthesis by Fréchet and Hawker developed
in 1990 (21). CuAAC (CuSO
4
, ascorbate, t-BuOH-H
2
O)
revealed to be a much more efficient methodology than
the originally reported nucleophilic substitution. Thus, not
only stoichiometric amounts of azide and alkyne partners
were employed, but also chromatographic purifications
were avoided in most of the cases. Shortly after, a comple-
mentary CuAAC-based divergent synthesis of dendrimers
was reported by the groups of Hawker and Wooley (22).
Mutifunctionalized dendrimers represent one of the most
interesting dendritic architectures for drug delivery. They
offer the possibility of selectively localizing diverse functional
molecules (e.g., drugs, targeting ligands, imaging agents)
while exploiting their inherent multivalency. In this regard,
a collaboration between the groups of Finn, Fokin, Sharp-
less, and Hawker demonstrated the usefulness of CuAAC for
the preparation of Janus-type dendrimers, where protected
and unprotected 2,2-bis(hydroxymethyl)propionic acid (bis-
MPA) dendrons were connected through their focal points
via triazole linkages [Cu(PPh
3
)
3
Br, DIPEA, THF, 50°C]
(Fig. 2a)(23). In addition, an adequate selection of protect-
ing groups allowed the stepwise CuAAC functionalization of
the resulting dendrimers (CuSO
4
, ascorbate, THF-H
2
O)
with 16 mannose units on one side of the dendrimer, and
2 coumarin dyes on the other. The resulting glycodendrimer
showed a 240-fold increase affinity towards lectin Conca-
navalin A (ConA) in hemagglutination experiments com-
pared to monomeric mannose which entails a rich future
for these structures in antiadhesive therapy. In a similar
fashion, the group of Sanyal has reported the preparation
of bifunctional Janus-type dendrimers based on a Diels-Alder
cycloaddition (benzene, 85°C) between furan-functionalized
Fréchet dendrons and maleimide-functionalized bis-MPA
dendrons (Fig. 2b)(24).
Another interesting approach for the selective localiza-
tion of various functionalities in dendrimers relies on the use
of repeating units of the type AB
2
C carrying three orthog-
onal handles. As described by Malkoch and coworkers, this
strategy has allowed the preparation of bifunctional
dendrimers of generation 1–3(G1-G3)bearingupto
24 hydroxyl groups at the periphery and 21 internal alkyne/
azide groups distributed throughout the dendrimer backbone,
which were amenable for further functionalization by means
of CuAAC (Fig. 2c)(25).
Since the synthesis of dendrimers demands a high degree
of control over activation and growing steps, chemists have
found in this process an excellent test bank for the assess-
ment of emerging click technologies. This has been the case
for example for the thiol-ene coupling (TEC), firstly
exploited by Hawker and coworkers in the dendrimer arena
starting from a tris-alkene triazine core and 1-thioglycerol
(26). Reactions were carried out in bulk (UV light, 365 nm)
without the need of removal of oxygen, and required only
1.5 equiv of thiol partner. The absence of byproducts
allowed easy purifications by precipitation up to G4. In a
similar fashion, the group of Stenzel has reported the
preparation of related thio-ether dendrimers by means
of thiol-yne coupling (TYC) (UV light, 365 nm, DMF)
(27). Interestingly, as alkynes allow the addition of two
thiols, dendrimers with a higher degree of functionaliza-
tion could be readily prepared through this approach.
This way, three generations of thio-ether dendrimers
peripherally decorated with carboxylic acids were pre-
pared and their potential in drug delivery illustrated by
complexation to cis-dichlorodiamineplatinum(II).
Hawker, Albertazzi, and coworkers have more recently
reported a combination of nucleophilic ring-opening of
904 Lallana et al.
epoxides by amines and TEC (UV light, 365 nm, MeOH) or
TYC [DMPA (2,2-dimethoxy-2-phenylacetophenone),
MeOH, UV light; or AIBN, MeOH, 80°C] as a useful
methodology for the preparation of dendrimers and PEG-
dendritic block copolymers internally functionalized with
hydroxyl groups (28,29). These examples are unique in the
application of two different click reactions for both the
activation and growing steps. The covalent attachment of
hydrophobic drugs/imaging agents at these internal hy-
droxyl groups was envisaged as an effective way to
provide conjugates with solubility and biocompatibility
properties similar to those of the unconjugated dendritic
scaffold.
As seen in Fig. 3, click chemistry has also revealed very
useful for the accelerated preparation of dendrimers from
repeating units carrying orthogonal functionalities. Such a
strategy results in a significant reduction of synthetic effort
since intermediate activation steps are avoided and hence,
every step translates into a new dendrimer generation. A
pioneering example in this field was reported back in 2001
by the group of Caminade and Majoral for the synthesis of
phosphorous-dendrimers involving hydrazone linkages in
one of the growing steps (Fig. 3)(30,31). More recently,
similar strategies have been implemented by the groups of
Hawker and Malkoch for the preparation of benzyl ether and
bis-MPA dendrimers combining CuAAC (CuSO
4
, ascorbate,
THF-H
2
O, 40°C) and etherification/esterification steps
(Fig. 3)(32). It has not been until recently, however, that two
different click reactions have been combined for this purpose
by the group of Kakkar [CuAAC (CuSO
4
, ascorbate, THF-
H
2
O, microwaves, 65°C) and Diels-Alder (EtOAc, 50°C)]
(Fig. 3)(33). This principle has been further pushed to the
limit by the groups of Malkoch and Hawker by synthesizing a
6th generation dendrimer in a single day by combination of
TEC (UV light, 365 nm, THF) and CuAAC (CuSO
4
,
ascorbate, THF-H
2
O) reactions (Fig. 3)(34).
Fig. 2 Representative examples of bifunctional dendrimers. Adapted with permission from (a) ref. (23); (b) ref. (24); and (c) ref. (25).
Clicking Polymers for Drug Delivery 905
Periphery Functionalization
As a consequence of their branched structure, dendrimers
display a high number of functional groups at their periph-
ery, which strongly determine their solubility and biological
properties. The development of efficient strategies for their
peripheral functionalization is therefore of great interest for
advanced drug delivery applications. The group of Hawker
showed in 2005 the usefulness of CuAAC for this purpose by
the efficient functionalization of various alkynated dendritic
scaffolds (benzyl ether, bis-MPA, PPI) with a small library of
azides (35). As opposed to traditional functionalization pro-
cedures, only a slight excess of reactive azides was necessary
under the reported conditions, being high dilution the only
required precaution to avoid acetylene homocoupling.
CuAAC Functionalization with Carbohydrates
The high versatility of CuAAC has been exploited for the
decoration of dendrimers with unprotected ligands of rele-
vance in drug delivery. Carbohydrates regulate a myriad of
biological and pathological processes in Nature. Recogni-
tion events such as fertilization, pathogen invasion, toxin
and hormone mediation, and cell-cell interactions rely on
multivalent carbohydrate-receptor interactions (36). This
cluster glycoside effect has prompted the development of
glycodendrimers and other synthetic multivalent glycocon-
jugates with the ability to interact with target lectins and
hence, to promote/inhibit natural carbohydrate-receptor
interactions (37). Pioneering examples on the preparation
of glycodendrimers from unprotected carbohydrates came
from the groups of Liskamp/Pieters (38), Finn/Fokin/
Sharpless/Hawker (23)(Fig.2a), and Fernandez-Megia/
Riguera (Fig. 4)(39,40). It is worth to note that, while the
first two reports relied on alkynated dendrimers, the group
of Fernandez-Megia and Riguera employed gallic acid-
triethylene glycol (GATG) dendrimers incorporating termi-
nal azide groups on their periphery. This way, glyco-
dendrimers and PEG-dendritic block copolymers were effi-
ciently prepared while ruling out the possibility of den-
drimer dimerization. Typical reaction conditions involved
a small excess of alkynated carbohydrates (CuSO
4
, ascor-
bate, t-BuOH-H
2
O) and isolation of the resulting function-
alized dendrimers by ultrafiltration. The study of the
interaction of these glycodendrimers with lectins by means
of surface plasmon resonance (SPR) shed valuable informa-
tion on the interpretation of multivalent carbohydrate rec-
ognition, highlighting the importance of the density of lectin
clusters on biological surfaces as a potential source of selec-
tivity in drug delivery (Fig. 4)(41).
CuAAC Functionalization with Peptides
Peptides are another interesting class of ligands that have
been exploited for the functionalization of dendrimers.
Peptide-dendrimer conjugates constitute valuable tools for
the analysis of a variety of multivalent processes such as
bacteria adhesion, cell proliferation, and allergic responses.
For instance, Arg-Gly-Asp (RGD) tripeptide and cyclo(Arg-
Gly-Asp-D-Phe-Val) [c(RGDfV)] pentapeptide are known
to bind integrin α
v
β
3
, a membrane protein that plays an
important role in tumor angiogenesis and metastasis (42).
The group of Liskamp has investigated the CuAAC conju-
gation of RGD and c(RGDfV) peptides to dendrimers
(CuSO
4
, ascorbate, microwaves, THF-H
2
O, 100°C) and
studied their increased affinity towards the α
v
β
3
integrin
Fig. 3 Repeating units employed in the accelerated preparation of dendrimers: Adapted with permission from (a) ref. (30,31); (b) ref. (32); (c) ref. (33);
and (d) ref. (34).
906 Lallana et al.
receptor (43,44). Functionalization with peptides proved to
be more troublesome than with carbohydrates (38), as very
low yields were obtained for the final conjugates in many
cases. The same authors have also found that
111
In-labeled
RGD-dendrimers specifically exhibited an enhanced in vivo
uptake into integrin α
v
β
3
overexpressing tumors (44). Other
biologically relevant peptides, such as antimicrobial magai-
nin I and II, or Leu-enkephalin, have been successfully
attached to the surface of dendrimers using similar CuAAC
strategies (45).
CuAAC Functionalization with Nucleotides
Nucleotide ligands have been also installed on the surface of
dendrimers. For instance, the group of Jacobson has recent-
ly decorated the surface of PAMAM dendrimers with nu-
cleotide antagonists of the P2Y
1
receptor by means of
CuAAC (CuSO
4
, ascorbate, THF-H
2
O) in order to inhibit
ADP-induced platelet aggregation (46). Interestingly, al-
though no significant multivalent effect was observed, the
affinity of these triazol-containing derivatives was higher
than the corresponding amide-linked conjugates, indicating
a positive effect of the triazole linker on activity. In a related
work, the same research group has combined CuAAC
(CuSO
4
, ascorbate, t-BuOH-H
2
O) and amide bond forma-
tion to prepare novel PAMAM conjugates containing ago-
nists of the P2Y
14
and the antiinflammatory A
3
adenosine
receptors (47).
CuAAC Functionalization with non-Naturally Occurring
Molecules
CuAAC has been also used to attach non-naturally occur-
ring molecules to the periphery of dendrimers. From a drug
delivery perspective, ionic residues are interesting ligands
that can lead to useful applications when presented onto
multivalent scaffolds. For instance, cationic ligands are
known to bind nucleic acids and result into gene delivery
vehicles when multivalently displayed onto polymers and
dendrimers (48). On the other hand, anionic polymers have
been described as mimetics of glycosaminoglycans with
applications in cancer therapy, as antiinflamatory agents,
or inhibitors of amyloid aggregation (49). In addition, poly-
ionic species have been used in the preparation of electro-
static complexes such as, micelles and assemblies with
significant impact in biotechnology and drug delivery (50).
In this context, the group of Fernandez-Megia and Riguera
has reported the synthesis of anionic dendrimers by means
t-BuOH-H2O
CuSO4, Sodium Ascorbate
CuAAC
N3n
O
HO
HO
OH
O
OH
-D-Mannoside
O
O
OH
OH
OH
-L-Fucoside
O
HO
HO
OH
O
OH
O
HO
OH
O
OH
-D-Lactoside
1nM
1M
KD~100 M
Lectin Lectin Lectin Lectin
Lectin Lectin
N
N
N
O
O
n
OH
μμ
ααβ
Fig. 4 CuAAC decoration of dendrimers with unprotected carbohydrates and increased affinity in multivalent carbohydrate recognition as a function of
generation and lectin density. Adapted with permission from ref. (39) and (41).
Clicking Polymers for Drug Delivery 907
of CuAAC (51). In a similar fashion to their previously
described work on glycodendrimers (39,40), azide-
decorated GATG dendrimers and PEG-b-GATG block
copolymers were functionalized under aqueous conditions
with sulfated, sulfonated, and carboxylated alkynes (CuSO
4
,
ascorbate, t-BuOH-H
2
O). This CuAAC strategy was
revealed more efficient than traditional coupling methods
(such as amide and sulfation procedures) which usually
result in non-homogeneous decoration patterns. Interesting-
ly, incubation of a sulfated PEG-b-GATG copolymer of G3
with equimolecular amounts of an oppositely charged poly-
L-Lysine (PLL) led to polyion complex (PIC) micelles as
potential drug delivery systems. These micelles showed an
improved stability compared to related systems from linear
block copolymers (51). Similar CuAAC conditions have
been more recently applied by Haag and coworkers for
the anionic functionalization of dendritic polyglycerols that
have been studied as L-selectin inhibitors (52).
In recent years, dendritic polymers carrying paramagnet-
ic ions have found application as contrast agents (CA) in
magnetic resonance imaging (MRI) (53). Particularly inter-
esting is the use of these macromolecular CA in quantitative
studies of microvessels and for prolonged angiographies,
both of interest in cancer diagnosis. From a synthetic point
of view, one of the major difficulties in the preparation of
dendritic CA for MRI is their complete surface functional-
ization with metal chelates, which often leads to mixtures of
compounds with varying degrees of substitution. This way,
not only the advantage of starting from monodisperse
materialsislost,butthefinalproductsbecomestrongly
batch-dependent. In this regard, the group of Fernandez-
Megia and Riguera has recently reported the advantage
of using CuAAC (CuSO
4
,ascorbate,t-BuOH-H
2
O) for
this goal by allowing the complete incorporation of pre-
formed Gd chelates onto the dendritic surface of PEG-b-
GATG block copolymers in very high yields (54). The
analysis of the physical and pharmacokinetic properties
in vitro and in vivo of this new family of dendritic CA
revealed them as a promising platform for the develop-
ment of CA for MRI.
The properties of PEG as antifouling agent to inhibit
protein adhesion as well as for increasing the aqueous solu-
bility and circulation times in the blood stream of covalently
bound molecules/nanosystems are well known (55). Shabat
and coworkers have reported the functionalization of self-
immolative dendrimers with PEG by means of CuAAC
(CuSO
4
, Cu wire, TBTA, DMF) to increase their water
solubility and prevent aggregation under aqueous condi-
tions (56,57). A PEGylated dendritic pro-drug of G2 with
four molecules of the anticancer agent camptothecin (CPT)
was prepared and the release of the drug demonstrated by
triggering with penicillin-G-amidase (Fig. 5). Cell-growth
inhibition assays demonstrated increased toxicity of the den-
dritic pro-drug upon incubation with the enzyme.
Fig. 5 Structure of a PEGylated (purple), self-immolative, dendritic CPT (blue) pro-drug with a trigger (red) designed for activation by penicillin-G-amidase.
Reproduced with permission from ref. (56).
908 Lallana et al.
Periphery Functionalization by Click Chemistries
other than CuAAC
The above examples highlight the impact and utility of
CuAAC in the synthesis and functionalization of den-
drimers. It must be pointed out, however, that in some
instances, Cu contamination might preclude the use of the
final conjugates in biological systems (12). This has been
illustrated by Weck and coworkers in the functionalization
of azide-decorated PAMAM dendrimers with an alkynated
PEG derivative (58). In this example, these authors demon-
strated that the use of CuSO
4
/ascorbate/t-BuOH-H
2
Oas
CuAAC coupling conditions resulted in dendrimers con-
taining up to 5000 ppm of Cu, despite extensive purification
protocols (extraction, dialysis, chromatography). The use of
the ligand BPDS along with CuI allowed the authors to
reduce the final Cu content down to 70 ppm, while substi-
tution of CuI for a Cu wire showed a further decrease to
only 40 ppm. In order to completely avoid Cu contamina-
tion in this case, the authors decided to move from CuAAC
to the Cu-free SPAAC alternative (11) by the use of a
cyclooctyne-PEG derivative.
To the best of our knowledge, no relevant examples on
the use of CuAAC for the conjugation of drugs to dendritic
scaffolds have been reported up to date. On the other hand,
alternative click reactions such as the formation of hydra-
zones and Michael addition are firmly established technol-
ogies for this goal. In a seminal report by the groups of
Fréchet and Szoka, these authors described the use of hydra-
zone linkages to conjugate doxorubicin (DOX) onto PEGy-
lated biodegradable Janus-type polyester (bow-tie)
dendrimers (Fig. 6)(59). Interestingly, the hydrazone linkage
not only provided a straightforward method for loading
DOX, but it also allowed its controlled release under the
acidic conditions at the endosome. Interestingly, while in
vitro experiments showed the DOX-dendrimer conjugate
to be 10 times less toxic than free DOX towards C-26
cultured cells, in vivo experiments in mice revealed improved
circulation half-life (31 h vs 10 min) and enhanced drug
uptake (9 times higher) for the conjugate. Also, efficacy
studies showed that a single intravenous injection of the
DOX-dendrimer conjugate (20 mg/kg DOX after 8 days
of tumor implantation) caused complete tumor regression
with a 100% survival of mice specimens over a 60-day
experiment, in a similar fashion to FDA-approved liposomal
drug carrier Doxil®. Additional advantages of this bow-tie
dendrimer conjugate are enhanced storage stability and ease
of formulation.
More recently, Haag and coworkers have relied on the
Michael addition of thiols to maleimides for the conjugation
of enzymatically cleavable anticancer pro-drugs onto den-
dritic polyglycerols (60). In this regard, self-immolative den-
drimers based on para-aminobenzyloxy-carbonyl coupled to
Phe-Lys dipeptide or to D-Ala-Phe-Lys tripeptide were pre-
pared and used for DOX and methotrexate conjugation,
respectively. The selected peptide linkers were chosen based
on their enzymatic cleavage by cathepsin B, a protease
overexpressed in several tumor types. Evaluation of these
dendrimer-drug conjugates revealed no beneficial effect for
DOX upon culture with pancreatic or mammalian carcino-
ma tumor cell lines (probably as a result of inefficient cellu-
lar uptake), but substantial improvement in antiproliferative
activity for the methotrexate-dendrimer conjugate.
SYNTHETIC POLYMERS
The concept of click chemistry has been defined by Sharp-
less and coworkers over three main lines (3): (a) the produc-
tion of properties through efficient transformations rather
Fig. 6 Functionalization of a PEGylated bow-tie dendrimer with DOX by means of hydrazone linkages. [PEO [poly(ethylenoxide)]; TFA (trifluoroacetic
acid)]. Adapted with permission from ref. (59).
Clicking Polymers for Drug Delivery 909
than challenging structures, (b) the use of readily available
starting materials, and (c) the final materials should be easy
to purify. Interestingly, these three same commandments
have also been the driving force of polymer science since
its very beginning. Indeed, polymer industry has prospered
by manufacturing functional materials from easily accessible
monomers using highly efficient chemical processes. It is not
surprising therefore that polymer scientists have embraced
the concept of click chemistry with enthusiasm, as reflected
by the large number of reviews dealing with polymers and
click procedures appeared in the literature since the original
click report (14).
In the following sections, attention will be focused on the
application of click chemistry for the synthesis and function-
alization of linear, star, and branched polymers of interest in
drug delivery. Examples dealing with the use of click proce-
dures with polymeric nanosized DDS (which arise from the
ability of polymers to aggregate in solution) have been
already surveyed in a previous Expert Review (8) and hence
are not covered herein.
Polymer Synthesis: Linear Polymers
The first examples of linear polymers prepared by CuAAC
were published by Fokin, Finn, and coworkers (61) and by
the group of van Maarseveen and Reek (62). These pioneer-
ing works within materials science (discovery of metal adhe-
sives and fluorene-based conjugated polymers, respectively)
were soon followed by a report from the group of Arora on
the preparation of peptidomimetic oligomers, which repre-
sent the first biologically relevant polymers prepared by
CuAAC (63). In this work, advantage was taken from the
planar and polarized structure of triazols to prepare
oligomers with similar properties to peptides but improved
in vivo stability. Since that seminal work, several other groups
have reported the preparation of a number of peptidomi-
metics by means of CuAAC (64).
Probably, the most important contribution to the field of
drug delivery using linear polymers synthesized by CuAAC
has been carried out by the group of Reineke (65–68). In
this series of reports, trehalose or cyclodextrin diazide
monomers have been copolymerized with linear oligoamine
monomers incorporating two terminal alkyne units (Fig. 7a).
While the presence of carbohydrates was envisioned to grant
biocompatibility, water solubility, and stability against ag-
gregation, the oligoamine monomers facilitated DNA com-
plexation and interaction with cell surfaces. Indeed, the
carbohydrate-oligoamine copolymers prepared this way
exhibited low cytotoxicity and facilitated a high cellular
uptake and gene expression in HeLa and H9c2(2-1) cells.
Alternative click reactions traditionally employed in the
preparation of synthetic polymers such as, Michael addition
or the ring-opening of epoxides, have recently found appli-
cation in interesting drug delivery programs. In an illustra-
tive example, Rege, Kane, and coworkers have prepared a
library of non-viral gene delivery vehicles via a combinato-
rial approach. By taking advantage of the clean reaction
between the epoxide groups at diglycidyl ethers and the
terminal amine groups at several linear oligoamines, a total
of eighty copolymers were synthesized in parallel (69)
(Fig. 7b). After primary screening and in vitro transfection,
a polymer with significantly higher transfection activity and
lower cytotoxicity than poly(ethylene imine) (PEI) could be
identified.
Fig. 7 (a) Linear carbohydrate-oligoamine polymers prepared by CuAAC as gene delivery vehicles. (b) Schematic representation of the preparation of a
library of cationic polymers prepared by ring-opening of diglycidyl ethers by primary amines. Adapted with permission from (a) ref. (65,67); and (b) ref. (69).
910 Lallana et al.
Side Chain Functionalization of Polymers
and Synthesis of Graft Copolymers
The ability to efficiently prepare polymers functionalized at
the side chains is considered of great importance in polymer
science. A typical approach to this goal involves the prepa-
ration of “specialized”monomers, as those bearing carbo-
hydrates or peptides of interest in biotechnology, for their
subsequent polymerization. However, such a strategy has
traditionally resulted troublesome or led to ill-defined mo-
nomeric species. In addition, the reactive functional groups
typically found in carbohydrates and peptides often result
incompatible with polymerization conditions or lead to un-
desired spontaneous polymerizations of the monomers. As a
result, it is not surprising that the incorporation of different
functionalities in a single polymer backbone has attracted
the interest of polymer chemists during the last decade.
An alternative strategy for the functionalization of the
side chains of polymers involves the polymerization of
monomers carrying adequate handles for subsequent func-
tionalization through efficient click processes. In this case,
the selection of the click reaction results crucial to avoid
again interference of the reactive handles with polymeriza-
tion mechanisms, as early illustrated by the group of Binder
when combining CuAAC and the ring-opening methathesis
polymerization (ROMP) (70). All attempts to polymerize an
alkynated 7-oxynorbornene derivative led to undesired
broad molecular weight distributions, probably due to com-
peting reactivity of the acetylene group with the ROMP
catalyst.
The first successful polymerization and subsequent
CuAAC side chain functionalization of a linear polymer
that was reported without the need of intermediate activa-
tion steps was published by Emrick and coworkers in 2005
(71). Copolymers prepared from ε-caprolactone (CL) and α-
propargyl-δ-valerolactone were successfully functionalized
with an azide-containing PEG and RGD peptide under
aqueous conditions (CuSO
4
, ascorbate, acetone-H
2
O, 80°C).
Interestingly, incubation of the resulting PEGylated graft
copolymer (5 mg/mL) with L929 mouse fibroblast showed
no qualitative change in cell monolayer morphology. In addi-
tion, the percentage of hemolysis of human red blood cells
induced by the graft copolymer was comparable to that of the
PEG precursor. Such results suggest a good biocompatibility
for these amphiphilic copolymers, and highlight the potential
of CuAAC for the functionalization of polymers for drug
delivery and other bioapplications.
In addition to the above example, a range of different
polymer backbones such as, polyoxazolines, polyisocya-
nides, and linear polypeptides among others, have been
successfully functionalized using CuAAC (6). Among these
polymers, those prepared via free radical polymerizations
have received the greatest impact by the application of
CuAAC. Pioneering work in this field has been done by
Matyjaszewski and coworkers, in which monomers contain-
ing acetylene and azido groups were polymerized via ATRP
(72). Interestingly, while poly(3-azidopropylmethacrylate)
could be prepared with good control over polymerization,
propargyl methacrylate (PMA) resulted in polymers with
high polydispersity, multimodal molecular weight distribu-
tions, and cross-linked networks, probably due to an undesired
participation of the alkyne group in the polymerization. In an
effort to solve this inconvenience, Haddleton and coworkers
decided to work with protected PMA monomers [3-(trime-
thylsilyl)propargyl (TMS-PMA)] which after polymerization
by ATRP, were deprotected and functionalized with α-
mannose and α-galactose azido derivatives by CuAAC [Cu
(PPh
3
)
3
Br, Et
3
N, DMSO] (73). The resulting glycopolymers
showed a strong affinity for Con A and Ricinus Communis
agglutinin lectins with affinities which depended on the den-
sity of the corresponding sugar, revealing the potential of this
synthetic approach for the preparation of libraries of biolog-
ically relevant glycopolymers.
The polymerization of monomers incorporating unpro-
tected alkyne groups via living radical polymerizations (LRP)
still remains elusive, with very few examples reported in the
literature (74,75). A very elegant alternative to the use of
protecting groups was reported by Haddleton, Mantovani,
and coworkers in 2008 by means of a simultaneous
CuAAC/ATRP of PMA using CuBr/iminopyridine as cat-
alytic system for both reactions (Fig. 8a)(76). Interestingly,
these authors found that the relative kinetics of CuAAC and
ATRP could be tuned by the selection of the solvent, with
the cycloaddition proceeding much faster than the polymer-
ization in toluene or at equal rates in DMSO. In any case,
the polydispersity indexes of the resulting polymers were in
the range 1.1–1.3, in agreement with a controlled polymer-
ization. Among the ligands that were attached by CuAAC,
it is worth to mention an unprotected azide functionalized
mannose which reveals this approach with a promising
future in drug delivery and other bioapplications, especially
if aqueous reaction conditions were developed.
In a similar fashion, the group of Emrick has prepared
polymeric phosphorylcholine‐CPT conjugates via a one-pot
ATRP/CuAAC protocol (77). These authors observed that
under the conditions employed for the polymerization of
methacryloyloxyethyl phosphorylcholine (MAPC) and
TMS-PMA (CuBr, bipyridine, DMSO-MeOH), deprotec-
tion of the TMS groups occurred which allowed the in situ
CuAAC functionalization of the polymer with various
azido-functionalized CPT to yield statistic zwitterionic
copolymers (Fig. 8b). These ionic drug-polymer conjugates
showed excellent water solubility thanks to the unique prop-
erties of MAPC, with no aggregation being observed even at
CPT loadings up to 14% in weight. In addition, the authors
proved that the rate of drug release could be tailored by
Clicking Polymers for Drug Delivery 911
controlling the nature of the linker between the azide group
and the CPT core. Interestingly, although IC
50
values of
these conjugates were higher than native CPT, the fact that
the PMA-MAPC backbone showed no apparent toxicity
envisages this strategy as an efficient approach in the design
of water-soluble polymeric supports for the delivery of high-
ly hydrophobic drugs.
As mentioned above, over the past decades several click
reactions have been extensively employed for the side chain
functionalization of polymers. Among them, the Michael
addition of thiols to α,β-unsaturated systems and the forma-
tion of hydrazones have received special attention in the
drug delivery arena. As an exhaustive analysis of this
literature falls outside the scope of this Expert Review,
interested readers are referred to specialized reviews
(78,79). With regard to emerging click technologies,
TEC has recently attracted most of the efforts of polymer
chemists. The first successful example on the application
of TEC for the side chain functionalization of polymers
was reported by Schlaad and coworkers with polyoxazo-
lines (80). The polymerization of 2-(3-butenyl)-2-oxazoline
resulted mild enough to allow the complete preservation
of the alkene groups which were subsequently functional-
ized via TEC (UV light, 303 nm, THF-MeOH) in a very
efficient way.
The intrinsic orthogonality displayed by click reactions
can be exploited for the simultaneous decoration of poly-
mers with different molecules of interest. This strategy pio-
neered by the group of Hawker in 2005 was initially tested
for the dual functionalization of polymers via CuAAC and
esterification reactions [CuBr(PPh
3
)
3
,DIPEA,THF]
(Fig. 9a)(81). Though the synthesis of esters does not comply
with the click philosophy, this first report illustrated the
advantage of combining precise chemistries in this field.
Application of the same principle was later reported by
Weck and coworkers for the simultaneous click functionali-
zation of a poly(norbornene) with a nucleoside and biotin by
means of CuAAC and hydrazone linkages (CuSO
4
, ascorbate,
DMF or DMSO, 25°C) (Fig. 9b)(82).
End Group Functionalization of Polymers
and Synthesis of Block Copolymers
End-functionalized polymers constitute the starting point for
the preparation of a wide variety of complex macromolec-
ular architectures, including block, miktoarm, dendritic, and
star polymers (83). Controlled living polymerizations allow
the easy introduction of functional end groups into poly-
mers, which have been exploited in subsequent click func-
tionalizations. Not surprisingly, in the first example of a
Fig. 8 Representative examples of CuAAC/ATRP protocols. Adapted with permission from (a) ref. (76); and (b) ref. (77).
912 Lallana et al.
clicked block copolymer, advantage was taken of the com-
bination of ATRP and CuAAC (84). The group of van
Hest showed in 2005 that the bromine group of PS
and poly(methyl methacrylate)(PMMA)preparedvia
ATRP could be easily transformed into azides by nu-
cleophilic substitution. CuAAC coupling of the resulting
polymers with one prepared from an alkynated ATRP
initiator (CuBr, DBU, THF) led to the expected block
copolymers (Fig. 10). This strategy allowed the prepa-
ration of a small library of block copolymers, some of
them from PEG-N
3
and PEG-alkyne derivatives. In all
cases, the formation of the block copolymer was shown
to proceed to completion by IR and SEC. More re-
cently, CuAAC has also been successfully combined to
other polymerization techniques such as reversible
addition-fragmentation chain transfer (RAFT) (85)and
ring-opening polymerizations (86).
The end group functionalization of linear polymers with
functional molecules can be used as a means to achieve new
properties. The first relevant example of this strategy with
CuAAC in drug delivery was reported by Lutz and cow-
orkers by functionalizing a poly(oligo(ethylene glycol) acry-
late) (POEGA) obtained by ATRP with a GGRGDG
hexapeptide (87). With this aim, an azide end group was
first installed in POEGA by nucleophilic substitution and
then reacted with an alkynated derivative of the hexapeptide
by means of CuAAC [CuBr, bipyridine, NMP] (Fig. 11a).
Unfortunately, no biological evaluation of the resulting con-
jugate was accomplished. In another interesting example,
the group of Kakuchi exploited the end decoration of an
azido-functionalized poly(N-isopropylacrylamide) (PNI-
PAM) with different small hydrophilic and hydrophobic
alkynated molecules by CuAAC (CuSO
4
, ascorbate, THF-
H
2
O) with the aim of tuning the characteristic lower critical
solution temperature (LCST) of PNIPAM (around 32°C,
depending on concentration and molecular weight)
(Fig. 11b)(88).
The combination of orthogonal click technologies for
the sequential functionalization of the end groups of het-
erotelechelic polymers appears as an attractive methodol-
ogy for the preparation of complex structures such as
triblocks and branched polymers. As a proof of concept,
the group of Hawker was able to sequentially modify a PS
bearing alkene and azide groups at the distal ends, by
means of CuAAC (CuBr, PMDETA, THF) and TEC
[UV light (365 nm), DMPA; or AIBN, 80°C] (89). Func-
tionalization was achieved starting from any of the end
groups, in agreement with a complete orthogonality of the
process (thermal thiol-ene conditions had to be employed
to avoid azide decomposition under UV irradiation). A
similar strategy based on Michael addition and TEC has been
more recently described (90).
Fig. 9 Multifunctionalization of linear polymers by simultaneous click reactions. [DMT (dimethoxytrityl)]. Adapted with permission from (a) ref. (81); and
(b) ref. (82).
Clicking Polymers for Drug Delivery 913
A further step towards the accelerated multifunction-
alization of linear polymers has involved the implemen-
tation of simultaneous processes. Up to date, two
protocols of this type have been reported for the prep-
aration of block copolymers. Hizal, Tunca, and cow-
orkers have described the combination of CuAAC and
Diels-Alder for the preparation of PEG-b-PS-b-PMMA
and PCL-b-PS-b-PMMA triblocks using a bifunctional
PS as central block carrying anthracene and azide
groups at the distal ends (CuBr, PMEDTA, DMF,
120°C) (Fig. 12)(91). Application of the same method-
ology has more recently allowed the same authors to
prepare a H-shaped quintopolymer from a central poly
(tertbutylacrylate) (92).
Preparation and Functionalization of Cyclic Polymers
One of the most challenging structures prepared via end
group modification of linear polymers is undoubtedly that
of cyclic polymers. The interest on cyclic polymers comes
not only from their challenging structures, but also from
their properties which are strongly influenced by that par-
ticular architecture. For instance, Szoka and coworkers
showed that cyclic polymers have characteristic pharmaco-
kinetic properties (93). Cyclic polyesters were prepared by
copolymerization of α-cholo-ε-caprolactone (α-Cl-ε-CL)
and ε-CL, so that azide could be easily introduced at a later
stage by nucleophilic displacement of the chlorine atom.
CuAAC (CuI, Et
3
N, THF, 40°C) was then used for the
Fig. 10 Preparation of linear
block copolymers by means of a
combination of ATRP and
CuAAC. Adapted with
permission from ref. (84).
Fig. 11 (a) End group functionalization of POEGA with a RGD peptide by means of CuAAC. (b) Synthesis and effect of end group on the LCSTof a library
of PNIPAM derivatives. Adapted with permission from (a) ref. (87) and (b) ref. (88).
914 Lallana et al.
functionalization of these polymers with alkynated PEG
chains (to improve water solubility) and a phenol derivative
(for radiolabeling with
125
I). It was revealed that cyclic
constructs displayed longer plasma circulation times than
the corresponding linear analogs, as the latter are able to
traverse more easily the nanoporous structure of kidneys.
Clearly, cyclic polymers represent promising structures
for the development of drug carriers with improved delivery
attributes. In this regard, great efforts have been recently
devoted for their preparation through click technologies.
The group of Matyjaszewski had already reported cyclic
PS as a side product during the step-growth CuAAC con-
densation of α-alkyne-ω-azide-PS (94). It was later demon-
strated by the group of Grayson that the concentration of
the polymer resulted to be a key factor for the preparation of
the cyclic construct (95). By employing a continuous addi-
tion technique, these authors avoided the utilization of high
dilution conditions, and cyclic PS up to 4200 Da could be
efficiently prepared. In a more recent contribution, Mon-
teiro and coworkers investigated the effect of polymer con-
centration and molecular weight, reaction temperature, feed
rate, and Cu concentration on this cyclization reaction (96).
An interesting alternative to the use of high dilution con-
ditions or slow addition processes, in Fig. 13 is depicted an
elegant preparation of cyclic polymers reported by the
group of Chen and Liu that takes advantage of the
unimer-micelle exchange equilibrium of thermo- and pH-
responsive polymers (97).
HYDROGELS
Hydrogels are hydrophilic and three-dimensional polymeric
networks capable of absorbing large amounts of water that
have found extensive application in drug delivery (98).
There are several examples on the use of click chemistry
for the synthesis and functionalization of hydrogels.
Michel Addition in the Preparation of Hydrogels
Probably, the first report on this field came out from the
group of Hubbel who used the Michael addition of thiols to
methacrylates for the cross-linking of hydrophilic PEG poly-
mers. The resulting gels were studied for the delivery of
Fig. 12 Schematic representation of a triblock copolymer synthesis by means of simultaneous CuAAC and Diels-Alder reactions. Adapted with permission
from ref. (91).
Fig. 13 Highly efficient
preparation of macrocyclic
diblock copolymers via
combination of supramolecular
self-assembly and intramolecular
CuAAC ring closure. Reproduced
with permission from ref. (97).
Clicking Polymers for Drug Delivery 915
proteins, with BSA taken as a model (99). Again by the use
of a Michael addition of thiols to vinyl sulfones, the group of
Segura has been able to prepare PEG-based hydrogels for
the delivery of DNA/PEI polyplexes to mesenchymal stem
cells (MSC). This gene delivery approach was envisaged to
promote the expression of tissue inductive factors locally
(Fig. 14)(100). Since MSC express high levels of matrix
metalloproteinase (MMP), MMP-degradable peptides were
used as cross-linkers to allow cell migration through proteo-
lytic degradation. In addition, RGD peptides were grafted
to PEG to promote cell adhesion.
Diels-Alder Cycloaddition in the Preparation
of Hydrogels
An interesting feature of the Diels-Alder reaction comes
from its reversibility at high temperatures, opening the door
to self-healing biomaterials (101). In the early 1990s, the
group of Saegusa described the use of furan and maleimide
end-functionalized poly(N-acetylethylimine) to prepare
hydrogels (CHCl
3
, room temperature) that could be gradu-
ally dissolved upon heating (MeOH-H
2
O, 80°C) (102). In a
related approach, Wei and coworkers have more recently
reported the preparation of hydrogels between furan-
functionalized dimethylacrylamide and bismaleimide-PEG
under aqueous conditions (103). These authors showed that
the resulting hydrogels were stable in water and that the
gelation time was strongly dependent on temperature,
which makes this an attractive approach for the preparation
of smart injectable materials.
CuAAC and SPAAC in the Preparation of Hydrogels
In recent years, the orthogonality and fast kinetics of
CuAAC have attracted the attention of the hydrogel com-
munity. The first example of the use of CuAAC for the
preparation of a hydrogel was reported by the group of
Hilborn in 2006 (104). Poly(vinyl alcohol) (PVA) was
modified either with azides or alkynes, producing two dif-
ferent polymers that yielded transparent hydrogels upon
mixinginthepresenceofCuSO
4
/ascorbate (Fig. 15a).
Low degree of functionalization of the PVA backbone was
required in order to maintain the water solubility of the
building blocks. Altogether, this intermolecular cross-linking
constitutes a promising alternative to traditional hydrogel
preparations involving bifunctional low-molecular weight
cross-linkers. As an example of this latter approach,
Hedrick, Hawker, and coworkers reported the preparation
of PEG-based hydrogels by CuAAC (Fig. 15b)(105). The
high efficiency on the cross-linking rendered hydrogels with
a more ideal structure (more even distribution of crosslink
junctions) that resulted in larger cavities and improved
properties (greater water adsorption, higher flexibility, en-
hanced tensile stress and tensile strain) compared to those
prepared by conventional approaches such as photochemi-
cal cross-linking.
One of the main problems associated to the preparation
of hydrogels via CuAAC is the toxicity of Cu. As a result,
thorough hydrogel purifications are required to ensure com-
plete removal of the metal catalyst. This critical purification
step might seem, however, like a contradiction when gela-
tion is performed in the presence of drugs (drug reservoir) or
aqueous suspensions of cells (tissue engineering scaffolds)
because of leaching. Illustrative examples in this field are
those reported by the groups of Crescenzi in 2007 (106) and
Dentini in 2009 (107) on the CuAAC mediated preparation
of hydrogels from hyaluronic acid. While control hydrogels
were extensively dialyzed against EDTA and water in order
to remove the Cu catalyst, no purifications were performed
when in the presence of model drugs (benzidamine and
DOX) or Saccharomices cerevisiae yeast cells.
As an alternative to the use of Cu as catalyst, more
benign alkyne-azide cycloaddition conditions for the prepa-
ration of gels have relied on the use of SPAAC. This Cu-free
strategy has been exploited by the group of Turro for the in
situ cross-linking of an azide-functionalized photodegradable
Fig. 14 Schematic
representation of the preparation
of PEG-based hydrogels
containing matrix metalloprotei-
nase sensitive peptides (MMPxl)
as linkers and their use for
the delivery of polyplexes to
MSC. Reproduced with
permission from ref. (100).
916 Lallana et al.
star polymer with linkers carrying two cyclooctynes
(Fig. 15c)(108). This strategy based on SPAAC was
expected not only to open the door to complex, functional,
and biocompatible networks, but also to allow better control
of gelation than CuAAC. In addition, the anaerobic con-
ditions required for similargelationsbasedonCuAAC
(109,110) were avoided in this case. Unfortunately, the
selection of poly(tertbutylacrylate) as model scaffold re-
quired gelation to be performed in organic media, with no
proof of drug delivery being accomplished (108). A step
forward in the SPAAC preparation of biocompatible hydro-
gels has been more recently made by the group of Anseth,
which has reported the preparation of PEG-based hydrogels
under physiological conditions for the in situ encapsulation
of 3T3 fibroblasts (111,112). In this case, the polymer net-
work was constructed from a star PEG tetraazide that was
cross-linked with a metalloproteinase cleavable peptide in-
corporating two terminal difluorocyclooctyne groups. In
addition, alkene groups were introduced in the peptide
backbone so that RGD peptides could be photopatterned
onto the scaffold using TEC, in order to show control over
cell growth. This way, two orthogonal and biocompatible
click reactions were combined for the preparation of highly
sophisticated materials mimicking the properties of the
extracellular matrix (Fig. 16).
CONCLUSIONS
The need of smart materials for the advance of drug deliv-
ery has resulted in polymers being designed for reducing the
toxicity, increasing the absorption, and improving the re-
lease profile of drugs. At the same time, the inherent multi-
valency of polymers and dendrimers has been exploited for
the incorporation of diverse functional molecules of interest
in targeting and diagnosis. Polymeric hydrogels with the
ability to encapsulate drugs and cells have also been devel-
oped for drug delivery and tissue engineering applications.
In this context, the introduction of the click concept by
Sharpless and coworkers in 2001, focusing the attention on
modularity and orthogonality, has greatly benefited poly-
mer synthesis, an area where reaction efficiency and product
purity are significantly challenged. Indeed, click chemistry
has revolutionized the synthesis of materials and refocused
chemist’s interests on the easy production of properties
rather than on challenging structures. The efficiency related
to click reactions has been embraced by researchers in
different fields and inspired the publication of hundreds of
papers in areas from materials and polymer science to
nanotechnology and drug delivery.
Although initially, the Cu(I)-catalyzed azide-alkyne cyclo-
addition (CuAAC) attracted most of the attention in the
field, many other reactions (some of them firmly established
before the click concept even being proposed) comply with
the click philosophy. For example, this is the case of the
Michael addition and Diels-Alder which have been tradi-
tionally used in drug delivery for decades. At the same time,
the introduction of the click concept has attracted renewed
interest on efficient classical transformations and the devel-
opment of new reactions, such as the thiol-ene (TEC) and
thiol-yne couplings (TYC).
Click chemistry has found in drug delivery an attractive test
bank for bioconjugation. The requirement of multifunction-
alized polymeric structures where selectively localizing drugs,
targeting ligands, and imaging agents in precise proportions
and sites has given the opportunity to assess the chemical
orthogonality between functional groups and to serve as
source of inspiration for the accelerated preparation/
functionalization of complex macromolecular architectures.
The possibility of clicking unprotected carbohydrates, pepti-
des, and nucleotides of interest in drug delivery onto polymers
and dendrimers has significantly accelerated the production of
properties while reducing synthetic efforts. In the same way,
click chemistry has given unprecedented access to complex
copolymers and dendrimers from stoichiometric amounts of
coupling partners. Cyclic polymers constitute another example
Fig. 15 Representative examples of CuAAC and SPAAC for the preparation of hydrogels. Adapted with permission from a) ref. (104); b) ref. (105); and c)
ref. (108).
Clicking Polymers for Drug Delivery 917
of the efficiency of click chemistry in drug delivery. They
represent promising structures for the creation of drug carriers
with long plasma circulation times that can be now prepared
with unprecedented fidelity thanks to click macrocyclization
reactions. In addition, the planar and polarized structure of
triazol linkages has been exploited for the preparation of
CuAAC-based peptidomimetics with similar properties to
peptides and improved in vivo stability. Although click reactions
have been traditionally employed for the fabrication of bio-
compatible hydrogels, it has not been until recently that vari-
ous orthogonal click reactions have been combined for the
simultaneous preparation/functionalization of highly sophisti-
cated hydrogels that mimic the properties of the extracellular
matrix. This continuous feedback between drug delivery and
Fig. 16 Macromolecular precursors react through SPAAC allowing for the direct encapsulation of cells within click hydrogels. The presence of terminal
alkenes in this three-dimensional network enables patterning of biological functionalities in real time and with micrometer-scale resolution by means of an
orthogonal thiol-ene photocoupling reaction (shown for three fluorescently labeled peptides using stereolithography). Adapted with permission from ref.
(111) and (112).
918 Lallana et al.
click chemistry is expected to accelerate current delivery
endeavors for the development of theranostics and complex
multimodal agents.
ACKNOWLEDGMENTS & DISCLOSURES
This work was financially supported by the Spanish Ministry
of Science and Innovation (CTQ2009-10963 and CTQ2009-
14146-C02-02) and the Xunta de Galicia (10CSA209021PR
and CN2011/037).
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