ARTICLE IN PRESS

Prog. Polym. Sci. 33 (2008) 448–477

www.elsevier.com/locate/ppolysci

The development of microgels/nanogels

for drug delivery applications
Jung Kwon Oha,, Ray Drumrighta,
Daniel J. Siegwartb, Krzysztof Matyjaszewskib,
a
The Dow Chemical Co., Dow Coating Solutions R&D Midland, MI 48664, USA
b
Department of Chemistry, Center for Macromolecular Engineering, Carnegie Mellon University, 4400 Fifth Avenue,
Pittsburgh, PA 15213, USA
Received 30 October 2007; received in revised form 22 January 2008; accepted 29 January 2008
Available online 12 February 2008

Abstract

Microgels/nanogels are crosslinked polymeric particles, which can be considered as hydrogels if they are
composed of water soluble/swellable polymer chains. They possess high water content, biocompatibility, and desirable
mechanical properties. They offer unique advantages for polymer-based drug delivery systems (DDS): a tunable size
from nanometers to micrometers, a large surface area for multivalent bioconjugation, and an interior network
for the incorporation of biomolecules. Present and future microgel applications require a high degree of control over
properties. They include stability for prolonged circulation in the blood stream, novel functionality for further
bioconjugation, controlled particle size with uniform diameter, and biodegradability for sustained release of drugs
for a desired period of time and facile removal of empty devices. This review describes the recent developments
of microgel/nanogel particles as drug delivery carriers for biological and biomedical applications. Various synthetic
strategies for the preparation of microgels/nanogels are detailed, including photolithographic and micromolding
methods, continuous microfluidics, modification of biopolymers, and heterogeneous free radical and controlled/living
radical polymerizations.
r 2008 Elsevier Ltd. All rights reserved.

Keywords: Microgels; Nanogels; Hydrogels; Drug delivery; Heterogeneous polymerization; Reverse micelles

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 449
2. Photolithographic and micromolding methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 451
2.1. Photolithographic techniques . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 451
2.2. Micromolding methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 452
3. Microfluidic preparation. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 453
4. Fabrication of biopolymers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 453

Corresponding authors.
E-mail addresses: jkoh2@dow.com (J.K. Oh), km3b@andrew.cmu.edu (K. Matyjaszewski).

0079-6700/$ - see front matter r 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.progpolymsci.2008.01.002
 ARTICLE IN PRESS
J.K. Oh et al. / Prog. Polym. Sci. 33 (2008) 448–477 449

4.1. Water-in-oil (W/O) heterogeneous emulsion methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 454

4.1.1. Inverse (mini)emulsion method . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 454
4.1.2. Reverse micellar method . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 456
4.1.3. Membrane emulsification. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 456
4.2. Aqueous homogeneous gelation method . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 457
4.3. Spray drying method. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 457
4.4. Chemical crosslinking of dextrans. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 458
4.4.1. Michael addition reaction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 458
4.4.2. Free radical polymerization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 459
5. Heterogeneous free radical polymerization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 460
5.1. Dispersion polymerization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 460
5.2. Precipitation polymerization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 460
5.3. Inverse (mini)emulsion polymerization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 462
5.4. Inverse microemulsion polymerization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 463
6. Heterogeneous controlled/living radical polymerization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 464
7. Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 466
Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 468
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 468

1. Introduction reversibly degrade into the corresponding precur-

The design and preparation of hydrogels have
attracted a great deal of interest in biomedical
engineering, pharmaceutical applications, and bio-
materials science because of their tunable chemical
and three-dimensional (3D) physical structure, good
mechanical properties, high water content, and
biocompatibility. These unique properties offer great
potential for the utilization of hydrogels in tissue
engineering, biomedical implants, drug delivery, and
bionanotechnology [1–5]. Recently, the development
of hydrogels hybridized with nano-sized materials
such as inorganic clays [6–9], carbon nanotubes
[10,11], and polyaniline nanosticks [12] has also been
of increasing interest in the field of materials science.
Hydrogels are generally prepared from hydro-
philic polymer matrices that are crosslinked by
several methods. One method is physical cross-
linking. Hydrogen bonds, crystallized domains,
hydrophobic interactions, stereocomplexation, tem-
perature-induced sol–gel transition, host–guest in-
teraction, aggregation, and self-assembly have been
utilized for the synthesis of ‘‘bulk hydrogels’’. They
include protein-based gels [13,14], polyelectrolyte-
based multilayered layer-by-layer (LbL) film hydro-
gels [15–17], triblock copolymer-based gelators
[18–21], polylactide (PLA)-based thermogelling
hydrogels [22–25], polyurethane-based hydrogels
[26], poly(acrylic acid-co-acrylamide) (P(AA-co-
AAm)) gels [27], hydrophobically modified hydro-
gels [28,29], organogels [30–33], and supramolecular
gels [34–38]. These physically crosslinked gels could
sors upon external stimuli.
Another method involves chemical crosslinking in
the presence of various crosslinkers. Several methods
have been explored for the preparation of effective
hydrogels. The modification of natural biopolymers
possessing a high degree of functional groups with
additional functional crosslinkers results in the
formation of biopolymer-based hydrogels [39–42].
Free radical polymerization (FRP) of water-soluble
or hydrophilic monomers in the presence of
either difunctional or multifunctional crosslinkers
allows for the preparation of synthetic hydrogels in
water. Various monomers have been examined,
including hydroxy-functional methacrylates [43,44],
poly((ethylene glycol) fumarate) [45], and (meth)a-
crylate derivatives of sugars [46,47], dendrimers [48],
poly(amino acid) [49], poly(ethylene glycol) (PEG)
[50], and PLA-b-PEG-b-PLA triblock copolymers
[51]. In particular, the FRP approach has been
extensively utilized to prepare stimuli-responsive
hydrogels, mainly based on poly(N-isopropylacryla-
mide) (PNIPAM). This is due to their unique
volume phase transition in response to external
stimuli such as temperature and pH [52–57].
Hydrogels can be in the form of macroscopic
networks or confined to smaller dimensions such as
microgels, which are crosslinked polymeric parti-
cles. When the size of microgels is in submicron
range, they are known as nanogels [58–60]. Micro-
gels/nanogels offer unique advantages for polymer-
based drug delivery systems (DDS) over polymer–
protein conjugates [61], polymer–drug conjugates
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Nomenclature PSS poly(sodium 4-styrenesulfonate)

PAH poly(allyamine hydrochloride )
PLA polylactide LbL layer-by-layer
DDS drug delivery systems PS polystyrene
MPS mononuclear phagocyte system PMMA poly(methyl methacrylate)
PDMS poly(dimethylsiloxane) PHEA poly(2-hydroxyethyl acrylate)
BSA bovin serum albumin PHPMA poly(2-hydroxypropyl methacrylate)
mTM microtransfer molding EGDMA ethylene glycol dimethacrylate
MIMIC micromolding in capillaries PNIPAM poly(N-isopropyl acrylamide)
PFPE perfluoropolyether LCST lower critical solution temperature
PEGDA poly(ethylene glycol diacrylate) DMIAAm dimethylmaleimidoethyl acrylamide
MeHA methacrylated hyaluronic acid PAA poly(acrylic acid)
HRP horseradish peroxidase DOX doxorubicin
SAMs self-assembled monolayers PAAm polyacrylamide
CS chitosan AETMAC 2-acryloxyethyltrimethylammonium
HA hyaluronan chloride
Dex dextran CMC critical micellar concentration
W/O water-in-oil PEO-b-PPO-b-PEO poly(ethylene glycol)-b-poly
ADH adipic dihydrazide (propylene glycol)-b-poly(ethylene
pDNA plasmid-DNA glycol)
EDCI ethyl-3-[3-dimethylamino]propyl OEGDMA oligo(ethylene glycol) dimethacrylate
carbodiimide PHEAS a,b-poly(N-2-hydroxyethyl)-D,L-aspar-
HAALD aldehyde-modified hyaluronan tamide
HAADH hydrazide-modified hyaluronan mTHPC meta-tetra(hydroxyphenyl)chlorine
SPG Shirasu porous glass AETMAC 2-acryloxyethyltrimethylaminium
DLS dynamic light scattering chloride
TPP tripolyphosphate CRP controlled/living radical polymeriza-
PEG poly(ethylene glycol) tion
PEI polyethyleneimine Mw/Mn molecular weight distribution
EDTAA ethylenediaminetetraacetic dianhydride PDI polydispersity
CTP camptothecin ATRP atom transfer radical polymerization
TEM transmission electron microscopy SFRP stable free radical polymerization
AFM atomic force microscopy RAFT reversible addition-fragmentation
Dex-SH thiol-functionalized dextrans chain transfer
HA-SH thiol-functionalized hyaluronans POEOMA poly(oligo(ethylene glycol)
GMA glycidyl methacrylate monomethyl ether methacrylate)
LA-HEMA hydroxy-terminated HEMA-lactate RES reticuloendothelial system
HEMA 2-hydroxyethyl methacrylate FITC fluorescein isothiocyanate
DMAEMA dimethylaminoethyl methacrylate RITC rhodamine B isothiocyanate

[62,63], micelles [64–66], and vesicles [67,68] based
on amphiphilic and doubly hydrophilic block
copolymers, dendrimers [69], and submicron-sized
particulates [70,71]. Microgels/nanogels have tun-
able size from nanometers to several micrometers
and a large surface area for multivalent bioconjuga-
tion [72]. They also have an interior network for the
incorporation of biorelated molecules. Physical
entrapment of bioactive molecules such as drugs,
proteins, carbohydrates, and DNA in the polymeric
network, as well as their in vitro release behavior,
have been extensively investigated as targeted drug
delivery carriers for biomedical applications. In
addition, the incorporation of inorganic nanocrys-
tals has been also reported, including quantum dots
[73,74] and magnetic nanoparticles [75,76] for
optical and magnetic imaging of living cells, and
gold nanorods for photodynamic therapy [77].
However, the present and future microgel appli-
cations still require a high degree of control over
 ARTICLE IN PRESS
J.K. Oh et al. / Prog. Polym. Sci. 33 (2008) 448–477
properties. Several criteria are required to design
and develop effective microgel-based DDS for
in vivo applications. One criterion is the stability
of microgels/nanogels for prolonged circulation in
the blood stream. This is important because the
instability of DDS may result in premature release
of therapeutics, causing adverse side effects. An-
other one is their novel functionality for further
bioconjugation of microgel surfaces with specific
ligands that can recognize receptors on diseased
cells. The effective ligands for cancer cells include
folic acid derivatives, peptides, proteins, and anti-
bodies [71,78,79]. Effective bioconjugation allows
for active targeting of microgels/nanogels to specific
cells, especially cancercells. Another criterion is
control over the dimension to be less than 200 nm
in diameter. This can facilitate cellular uptake of the
nanogels through a receptor-mediated endocytosis
to cross cell membranes as well as reduce nanopar-
ticle uptake by mononuclear phagocyte system
(MPS); consequently, this will significantly increase
their circulation time in blood [80]. The last criterion
is the biodegradability of microgels/nanogels. Bio-
degradation should not only modulate the release of
drugs for a desired period of time, but also enable
removal of the empty device after drug release. A
significant amount of work has been directed
toward the development of crosslinkers functiona-
lized with (bio)degradable linkages. These include
peptides [39,81–83], anhydrides [84], oligo(lactate)
esters [85–89], polyperoxides [90], disulfides [91,92],
acetals [93–95], poly(3-hydroxybutyrate) [96], and
polyphosphoesters [97,98]. The resulting hydrogels
crosslinked with these linkages were degraded to
water-soluble polymers in external environments.
For example, oligo(lactate) ester linkages were
hydrolyzed in physiological conditions at pH ¼ 7.0
at 37 1C. Disulfides were degraded into the corre-
sponding thiols in presence of water-soluble redu-
cing agents, including dithiothreitol [99,100] and a
tripeptide glutathione [101]. Acetals were degraded
under acidic conditions (pHo6.5), while they are
stable at physiological pH ¼ 7.4 [93].
This review will describe the recent developments
of microgel/nanogel particles as drug delivery
carriers for biological and biomedical applications.
Various synthetic strategies for the preparation of
microgels/nanogels will be presented. They include
photolithographic and micromolding methods, mi-
crofluidics, modification of biopolymers with var-
ious approaches, and free radical heterogeneous
polymerization in dispersion, precipitation, inverse
451
(mini)emulsion, and inverse microemulsion. In
addition, recent advance on controlled/living radical
polymerization (CRP) in heterogeneous media to
prepare well-defined microgels/nanogels with un-
ique properties for drug delivery will also be
discussed.
2. Photolithographic and micromolding methods
2.1. Photolithographic techniques
Soft- and photolithography [102–104] has been
explored to fabricate 3D hydrogel particles
[105,106] and microgel rings [107] for drug delivery
and tissue engineering. The photolithographic
method requires the development of techniques for
surface treatment of stamps or new materials for
replica molds to permit the release of molded gels
from stamps or replica molds. Poly(dimethylsilox-
ane) (PDMS) stamps have generally been used to
mold, release, and stack gels into 3D structures.
Surface modification of PDMS stamps with hex-
a(ethylene glycol)-terminated self-assembled mono-
layers (SAMs) or adsorbed monolayers of bovin
serum albumin (BSA) promoted the release of
molded gels to a substrate without distortion.
Replica molding, microtransfer molding (mTM),
micromolding in capillaries (MIMIC), and other
standard lithographic techniques were applied to
fabricate gels with various microstructures including
monolithic gels, isolated microstructures, and inter-
connected networks. In addition, various gel pre-
cursors were examined, such as protein-based
collagen, gelatin, and tumor extract Matrigel as
well as sugar-based agarose gel [105].
A top-down method called ‘‘Particle Replication
In Nonwetting Templates (PRINT)’’ [106] was
developed to fabricate submicron-sized microgels
with control over particle size, shape, and composi-
tion. A photocurable perfluoropolyether (PFPE)
replica was used as the molding material. This
eliminated the formation of a residual interconnect-
ing film between molded objects, thus allowing for
the preparation of isolated objects (Fig. 1). In the
approach, silicon master templates were mainly
made by using electron beam lithography. Dimeth-
acrylate-functionalized PFPE oligomers containing
a photoinitiator were poured onto various patterned
silicon master templates, and then photochemically
cured to form patterned elastomeric PFPE replica
mold. PFPE-based materials have many advantage-
ous properties including solvent resistance, chemical
 ARTICLE IN PRESS
452 J.K. Oh et al. / Prog. Polym. Sci. 33 (2008) 448–477
Fig. 1. Illustration of the PRINT process (upper) and TEM images of microgels of PEGDA with various shapes prepared using PRINT
(lower): trapezoidal (a), bar (b), conical (c), and arrow shape (d). Reprinted with permission from Ref. [106]. Copyright 2005 American
Chemical Society.
robustness, and durability. They have also been
used for different applications to fabricate organic
solvent resistant microfluidic devices with features
on the order of hundreds of microns [108,109].
Using PRINT, monodisperse microgels of poly
(ethylene glycol diacrylate) (PEGDA), triacrylate
resin, PLA, and polypyrrole were prepared. Their
sizes were ranged from 200 nm to micron-scale
in diameter with various shapes such as trapezoidal,
bar, conical, and arrow. In addition, DNA,
proteins, and small-molecule therapeutics were
incorporated into 200 nm PEG-based microgels
using a simple encapsulation technique to demon-
strate the compatibility of PRINT with (bio)
molecules.
2.2. Micromolding methods
Micromolding of hydrogels has been examined as
a potential method to fabricate submicron-sized
microgels. The methods are similar to photolitho-
graphic techniques. However, they can minimize the
need to use costly lithographic equipment and clean
room facilities. Moreover, by controlling the fea-
tures on a mold stamp, they enable control over the
size and shape of microgels, which are important for
biomedical applications.
The preparation of shape-controlled, harvestable
hydrogel particles encapsulated with mammalian
cells was demonstrated using micromolding method
[110]. In the process, cells were suspended in a
hydrogel precursor solution consisting of either
methacrylated hyaluronic acid (MeHA) or PEGDA
and a photoinitiator in water. The resulting mixture
was deposited onto plasma-cleaned hydrophilic
PDMS patterns and then photocrosslinked via
exposure to UV light. The resulting cell-laden
microgels were removed, hydrated, and then har-
vested. They were also molded into various shapes
including square prisms, disks, and strings.
A similar method was reported for the prepara-
tion of cell-laden chemically and physically cross-
linked hydrogel particles for tissue engineering
[111]. This approach utilized the controlled release
of gelling agents from the mold to enable rapid
gelling upon contact of hydrogel-forming precur-
sors with the gelling agent in the mold. Ionically
crosslinked hydrogels of alginate, a naturally
occurring carbohydrate-based polymer, were pre-
pared from crosslinking of alginates molded
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J.K. Oh et al. / Prog. Polym. Sci. 33 (2008) 448–477
between PDMS and calcium-containing agarose
slab upon the release of calcium ions from the slab.
The obtained microstructured hydrogel particles
had lateral dimensions between 5 and 2000 mm and
vertical dimensions between 10 and 200 mm.
3. Microfluidic preparation
Microfluidic methods have been recently explored
for the preparation of monodisperse micron-sized
microgels. The methods require the fabrication of
microfluidic devices by soft lithography using elasto-
meric materials, particularly PDMS or polyurethane
elastomers as building blocks. The devices generally
consist of inlets for monomers (or oligomers) and
continuous liquids, and microchannels with a tapered
junction where two immiscible phases are merged.
Emulsification of monomers by breaking up liquid
threads to droplets and in-situ crosslinking of the
resulting droplets by photopolymerization or poly-
condensation are the two general steps involved in the
continuous microfluidic preparation of microgels.
Confinement of droplets, variation of flow rates of
liquids, and precise control of reaction time are key
parameters to generate monodisperse particles with a
variety of shapes and morphologies [112]. More
recently, the microfluidic preparation of complicated
nanostructured particles such as Janus and ternary
particles has been reported [113].
Several approaches were examined for the micro-
fluidic preparation of micron-sized microgels of
both synthetic and biological polymers. They are
based on gelation methods in microchannels;
chemical gelation, physical gelation by temperature
change [114], reversible shear thinning [115], and
ionic crosslinking [116,117], and coalescence-in-
duced gelation [118]. In this review, chemical
gelation and ionic crosslinking will be discussed.
Details on other gelation methods have been
summarized in a mini-review [112].
Chemical gelation utilizes photoinduced polymer-
ization to crosslink low-viscosity monomer droplets
stably dispersed in a continuous phase with an aid
of surfactants. This method was mostly examined
for the preparation of microgels of synthetic
polymers. Examples include the preparation of
10-mm-sized monodisperse dextran (Dex)-modified
microgels [119] and photocrosslinked microgels
immobilized with biocatalysts such as glucose
oxidase (GOx) and horseradish peroxidase (HRP)
[120]. Physical gelation by ionic crosslinking can be
achieved in a microchannel using two approaches;
453
internal and external gelation. For the former,
droplets consist of a gelling polymer and a cross-
linking agent precursor in inactive form. Contin-
uous phase contains an activator that can diffuse
into droplets and react with crosslinking agent
precursors, triggering the release of crosslinking
agents. The released crosslinking agents can cause
gelation. For the latter, droplets are emulsified in
continuous phase that contains a crosslinking agent.
Diffusion of crosslinking agents to emulsified
droplets leads to gelation. As a consequence, the
crosslinking agent should be soluble in aqueous
droplet phase.
Recently, physical gelation by ionic crosslinking
using internal and external crosslinking approaches
was explored for the microfluidic preparation of
microgels of alginate crosslinked with calcium ions
(Ca2+) (Fig. 2) [112]. For internal gelation, an
aqueous sodium alginate dispersion containing
CaCO3 as a crosslinking agent precursor was
supplied from the central channel and soybean oil
containing acetic acid as an activator was supplied
from the side channels. Two liquid streams passed
through a tapered junction to form emulsified
droplets in soybean oil continuous phase. Acetic
acid that diffused to the aqueous droplets reacted
with CaCO3. The generated Ca2+ ions as cross-
linking agents involved in crosslinking reactions
with carboxylic acid residues of sodium alginate,
resulting in the formation of alginate microgel
particles. However, weak gelation was identified as
the most important shortcoming, along with poor
control over microgel morphology for the method.
For external gelation, aqueous sodium alginate
solution was emulsified in soybean oil containing
sodium acetate. Downstream of the channel, Ca2+
ions diffused to aqueous droplets to crosslink
alginate. Contrary to internal crosslinking, this
method resulted in the successful preparation of
stable monodisperse spherical alginate microgels.
Their sizes varied from 50 to 70 mm with the flow
rates of aqueous and organic phases. In addition,
control over the diffusion time of crosslinking agent
in aqueous phase and the concentration of cross-
linker in organic phase allowed for the preparation
of various alginate microgels with capsular, gradi-
ent, and uniform structures [121].
4. Fabrication of biopolymers
Chitosan (CS), hyaluronan (HA), and Dex are
naturally occurring carbohydrate-based biopolymers.
 ARTICLE IN PRESS
454 J.K. Oh et al. / Prog. Polym. Sci. 33 (2008) 448–477
Fig. 2. Illustration of the microfluidic production of alginate microgels by internal gelation (a) and external gelation (b). Reprinted with
permission from Ref. [112]. Copyright 2007 Wiley InterScience.
Fig. 3 shows their chemical structures. CS is a
polysaccharide composed of 2-amino-deoxy-D-glucan
combined with glycosidic b(1-4) linkages. It is
commercially obtained by hydrolysis of aminoacetyl
groups of naturally occurring chitin, which is the
main component of shells, crabs, shrimp, and krill
[122,123]. HA is a naturally occurring mucopoly-
saccharide composing of N-acetyl-D-glucosamine
and D-glucuronic acid, found in the extracellular
tissue matrix of vertebrates. Dex is a bacterial-
derived polysaccharide consisting of a-1,6-linked
D-glucopyranose residues, and generally produced
by enzymes from certain strains [124]. These
carbohydrate-based biopolymers are non-toxic and
offer high water solubility, biocompatibility, and
biodegradability. Due to their unique properties,
these biopolymers have been used for various
applications in biomedicine, pharmaceutics, materi-
als science, cosmetics, and food industry. Another
feature of these biopolymers is a high content of
functional groups, including amino groups in CS
and carboxylic acid groups in HA. These functional
groups can be utilized in crosslinking with addi-
tional functional crosslinkers, resulting in the
formation of functional microgels of biopolymers.
Furthermore, the residual functional groups can
also be utilized for further bioconjugation with cell-
targeting agents.
Many methods have been developed for the
preparation of microgels of these biopolymers.
They can be classified into four categories: water-
in-oil (W/O) heterogeneous emulsion, aqueous
homogeneous gelation, spray drying method, and
chemical crosslinking of Dex. For CS, the prepara-
tion of micro/nanoparticles as drug delivery carriers
has recently been reviewed [125].
4.1. Water-in-oil (W/O) heterogeneous
emulsion methods
W/O emulsion methods involve generally two
steps: emulsification of aqueous droplets of water-
soluble biopolymers in continuous oil phase with an
aid of oil-soluble surfactants and crosslinking of
biopolymers with water-soluble crosslinkers. The
general approaches include inverse (mini)emulsion
(often called emulsion crosslinking technique),
reverse micelle, and membrane emulsification.
4.1.1. Inverse (mini)emulsion method
A W/O emulsion is formed from a mixture
consisting of aqueous biopolymer droplets and a
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J.K. Oh et al. / Prog. Polym. Sci. 33 (2008) 448–477
continuous oil phase using either a homogenizer or
a high-speed mechanical stirrer. Mineral oil and
hexane as hydrophobic organic solvents and Span
80 (sorbitan monooleate) and Aerosol OT (sodium
bis(2-ethylhexyl) sulfosuccinate) as oil-soluble sur-
factants were often used to implement colloidal
Fig. 3. Chemical structures of repeating unit of chitosan,
hyaluronan, and dextran.
Fig. 4. Illustration of crosslinking reaction of hyaluronan with adipic dihydrazide [126].
455
stability of the resulting inverse emulsion. Various
drugs, DNA, and cells are physically incorporated
into aqueous droplets. The resulting aqueous
droplets of biopolymers are then crosslinked with
appropriate crosslinking agents. The resulting cross-
linked microgel particles are prepared as dispersion
in organic solvents, and thus purified by precipita-
tion, centrifugation, washing with organic solvents
such as isopropanol, and lyophilization. By this
method, the size of the prepared microgel particles
can be controlled by amount of surfactants and
crosslinking agents as well as stirring speed during
the formation of inverse emulsion.
For preparation of HA-based microgels, car-
boxylic acids of HA were crosslinked with adipic
dihydrazide (ADH) as a crosslinker in the presence
of ethyl-3-[3-dimethylamino]propyl carbodiimide
(EDCI) in aqueous droplets (Fig. 4). Plasmid-
DNA (pDNA) was then incorporated into the
robust microspheres with a size distribution of
5–20 mm for sustained gene delivery and site-specific
targeting application [126]. In another case, HA was
chemically modified with aldehyde functionalities
generated from vicinal secondary alcohols on HA
by oxidation with sodium periodate, resulting
in the formation of aldehyde-modified hyaluronan
(HAALD). HA was also modified with dihydrazide
functionalities generated from carboxylic acids on
HA by coupling with glutahydrazide, forming
hydrazide-modified hyaluronan (HAADH). The
resultant HAALD and HAADH were homogenized
in mineral oil with Span 80 to form microspheres
with a size of 10–15 mm for vocal fold regeneration
[127]. The microgels were crosslinked with other
reactive polymers to form doubly crosslinked
 ARTICLE IN PRESS
456 J.K. Oh et al. / Prog. Polym. Sci. 33 (2008) 448–477
networks that exhibited elastic moduli similar to
those of vocal fold lamina propria at frequencies
close to the range of human phonation.
For preparation of CS-based microgels, glutar-
aldehyde has been mainly used as an effective
crosslinker that reacts with amino groups on CS in
aqueous droplets. Various drugs including diclofe-
nac sodium, 5-fluorouracil, phenobarbitone, theo-
phylline, pentazocine, and progesterone were added
into aqueous droplets [128–133]. The resulting drug-
loaded CS-based microspheres had a relatively large
size ranging from 40 to 300 mm. A similar approach
has been applied to prepare complex microgels by
coacervation of CS glutamates and HAs without
external crosslinkers in mineral oil containing Span
80. Gentamicin sulfate as model drug was incorpo-
rated into microgels to examine its in vitro release
profile [134].
4.1.2. Reverse micellar method
Similar to the inverse (mini)emulsion method, the
reverse micellar method also involves a W/O
dispersion; however, a relatively large amount of
oil-soluble surfactants is used to form a thermo-
dynamically stable micellar solution consisting of
aqueous droplets dispersed in the continuous oil
phase [135,136]. The resulting micellar droplets have
a submicron size ranged from tens to hundreds of
nanometers in diameter (Fig. 5).
Tumor targeted CS-based nanogels were pre-
pared in inverse microemulsion of hexane contain-
ing Aerosol OT as a stabilizer in the presence of
doxorubicin (Dox)-modified Dex. Aqueous glutar-
aldehyde was used to crosslink CS. The resulting
Dox-encapsulating CS-based nanogels have a dia-
meter of around 100 nm [137]. HA-based nanogels
were also prepared in hexane containing a surfac-
tant mixture of Span 65, Span 80, and Tween 80.
This approach involves the modification of HA to
corresponding thiol-functionalized HA (HA-SH)
Fig. 5. Illustration of the reverse micellar method for the preparation of nanogels.
through a carbodiimide coupling reaction with
cystamine at pH ¼ 7.0 and then thiol-disulfide
exchange in the presence of dithiothreitol at
pH ¼ 8.0. The resulting thiol-HA was crosslinked
in reverse micelles containing small interfering RNA
(siRNA) by reduction of thiols to disulfides to form
nanogels with a diameter of 198 nm size for target-
specific intracellular delivery of siRNA [138].
4.1.3. Membrane emulsification
Membrane emulsification is a relatively new
method for the preparation of spherical particles
with a highly uniform size distribution [139]. The
method involves the use of a membrane, initially
Shirasu porous glass (SPG) membrane with a highly
uniform pore size ranging from 0.1 to 18 mm, through
which a liquid is allowed to permeate under adequate
pressure [140]. The resulting uniform-sized droplets
are dispersed in the continuous phase, resulting in the
formation of simple W/O and O/W emulsions,
multiple emulsions such as O/W/O, O/W/O, and
solid/O/W dispersions, and the corresponding parti-
cles with various shapes including microspheres,
hollow spheres, core–shell microcapsules, and orga-
nic–inorganic hybrid materials. Details on such
methods using membrane emulsification are sum-
marized in several review papers [141–144].
This technique combined with a step-wise cross-
linking method was recently developed to prepare
microgel particles with a uniform size distribution
[145]. Fig. 6 illustrates the preparation process of CS
microspheres containing insulin. First, a stable W/O
emulsion with a uniform droplet size was formed by
passing an aqueous CS/acetic acid solution contain-
ing insulin through the uniform pores of a porous
glass membrane into a paraffin/petroleum ether
mixture containing hexaglycerin pentaester (PO-500)
emulsifier. Next, the uniform droplets of CS were
hardened by both ionic crosslinking with tripoly-
phosphate (TPP) and chemical crosslinking with
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J.K. Oh et al. / Prog. Polym. Sci. 33 (2008) 448–477
Fig. 6. Illustration of a process for the preparation of chitosan microspheres containing insulin by a membrane emulsification technique
combined with a step-wise crosslinking method. Reprinted with permission from Ref. [145]. Copyright 2006 Elsevier.
glutaraldehyde. The solidification condition was opti-
mized based on microsphere morphology, encapsula-
tion efficiency, drug activity, and release profile
in vitro. The resulting uniformly sized insulin-loaded
microgels had a size range from 4 to 15 mm in
diameter. A similar method was applied to prepare
CS-based microgels incorporated with BSA [146] and,
more recently, agarose beads with different particle
size ranging from 15 to 60 mm [147].
4.2. Aqueous homogeneous gelation method
Covalent chemical crosslinking was utilized for
the preparation of biopolymer-based nanoparticles
in water. An example includes the utilization of a
facile carbodiimide coupling reaction of CS with
a PEG dicarboxylic acid as a water-soluble cross-
linker, resulting in the formation of CS-based
nanogels with a diameter of 4–24 nm by transmission
electron microscopy (TEM) and 50–120 nm
by dynamic light scattering (DLS) [148]. In another
example, ethylenediaminetetraacetic dianhydride
(EDTAA) was used to react with CS for the
preparation of pH-sensitive nanogels with a dia-
meter of 70–80 nm. They enabled reversible switch of
their surface upon pH change to be positive
at pHo4.8 and negative at pH45.2, and were
stable in the entire pH range. These characteristics
suggest that they are an effective candidate for
encapsulation of pH-sensitive anticancer agents such
as highly water-insoluble camptothecin (CPT) [149].
Reversible physical crosslinking has also been
explored to prepare fine nanoparticles. In particu-
lar, ionic gelation involves electrostatic interaction
of cationic CS with polyanion, polyethyleneimine
(PEI) [150], and typically TPP [151–153] in water.
The method is advantageous in that it avoids the
457
possible toxicity of crosslinkers and reagents that
are involved in chemical crosslinking method. For
the development of drug delivery carriers to target
tumors, CS was recently modified with glycidyltri-
methylammonium chloride to form N-[(2-hydroxy-
3-trimethylammonium)propyl]chitosan chloride,
which was in turn crosslinked with TPP in the
presence of methotrexate, an anticancer drug.
The resulting CS-nanogels underwent a change in
volume upon decreasing pH, varying the diameter
from 180 nm at pH ¼ 7.4 to 450 nm at pH ¼ 5.0.
This volume transition is effective for targeted drug
delivery of cancer therapeutics in that methotrexates
are physically entrapped in CS-based nanogels at
physiological pH ¼ 7.4, while they are released
from the nanogels in tumor cells under acidic
conditions. In addition, these nanogels were con-
jugated with transferrin, a cell-targeting protein, in
aqueous media, which facilitates receptor-medicated
endocytosis. The resulting methotrexate-loaded CSs
nanogel bioconjugates could be used for targeted
pH-mediated intracellular release of cancer thera-
peutics (Fig. 7) [154,155].
4.3. Spray drying method
Spray drying is widely used in pharmaceutical and
materials science to prepare capsules, granules, fine
powders, and agglomerates. This method involves
the use of a spray dryer, mainly consisting of
atomizer and drying chamber. Solutions and suspen-
sions of drugs, polymers, and particles are atomized
to fine droplets. A stream of hot air induces quick
evaporation of solvent from the droplets in drying
chamber, resulting in the formation of microspheres
or microgels. The obtained particles settle into a
bottom collector, which are further dried in a
 ARTICLE IN PRESS
458 J.K. Oh et al. / Prog. Polym. Sci. 33 (2008) 448–477
Fig. 7. A schematic illustration of the preparation of chitosan-based microgels using ionic gelation (a), bioconjugation (b), loading (c), and
release of drugs (d) for drug delivery targeted to tumors. Reprinted with permission from Ref. [155]. Copyright 2006 American Chemical
Society.
vacuum chamber or modified in separated experi-
ments. The size of the resulting microspheres is
determined by nozzle size, spray flow rate, atomiza-
tion speed, and extent of crosslinking.
This method has been explored to prepare drug-
loaded microspheres of CS [156–160] and HA [161]
as biodegradable drug delivery carriers. An aqueous
solution of these biopolymers containing cross-
linkers was spray-dried with various drugs, such as
cimetidine, sodium dichofenac, vitamin D2, ampi-
cillin, betamethasone disodium phosphate, and
cromolyn sodium salt. The resulting spheres had a
size of 1–10 mm in diameter. Further encapsulation
of the resultant biomicrospheres was achieved. As
an example, CS-based microspheres with a size of
1.8–2.9 mm were prepared by spray drying method,
and then encapsulated into Eudragits microspheres
with a size ranging from 152 to 223 mm using an
oil-in-oil solvent evaporation method [162].
The spray drying technique has also been used
to prepare submicron-sized nanoparticles of silica/
poly(L-lysine)/alginate [163]. In-situ reduction of
Co2+ ions resulted in the formation of magnetic
cobalt silicate nanoparticles. The resulting nano-
particles could enter cells through endocytosis and
degrade in fibroblast cells, suggesting that they may
be suitable for targeted drug delivery applications.
4.4. Chemical crosslinking of dextrans
Biodegradable Dex-based microgels and hydro-
gels were prepared by various methods based on
chemical crosslinking, including carbodiimide cou-
pling [42], Michael addition, and free radical
polymerization. The next section will detail Michael
addition and free radical polymerization. Physical
crosslinking based on stereocomplexation and self-
assembly was also reported for the preparation of
nanogels [164,165].
4.4.1. Michael addition reaction
A Michael addition-type reaction has been
explored to prepare hydrogels with thiol-acrylate
networks. This method requires the modification of
hydroxy groups of Dex with either thiols or
acrylates [166]. Thiol-functionalized Dex (Dex-SH)
was prepared by a two-step reaction: activation of
hydroxy groups of Dex with 4-nitrophenylchloro-
formate and then reaction of the resultant nitro-
phenyl-substituted Dex (Dex-NP) with cysteamine
(Fig. 8). The thiol-Dex was then added into a PEG
tetra-acrylate, resulting in the formation of Dex-
based hydrogels [167]. In another case, vinylsulfone
functionalized Dex (Dex-VS) was prepared by
reaction of Dex with vinyl sulfone alkanionic acid,
which was prepared by the reaction of a mercapto-
alkanoic acid with divinyl sulfone (Fig. 8). They
were crosslinked with linear and four-arm star
mercaptopoly(ethylene glycol), which was prepared
by multi-step organic synthesis, to form Dex-
hydrogels [168].
Michael addition reaction was also utilized for
the preparation of HA-based hydrogels. In this case,
HA-SH was prepared by reaction of carboxylic
 ARTICLE IN PRESS
J.K. Oh et al. / Prog. Polym. Sci. 33 (2008) 448–477 459

Fig. 8. Schematic representation for the synthesis of thiol-functionalized dextran (Dex-SH) [167] and vinylsulfone-functionalized dextran
(Dex-VS) [168].

Fig. 9. Illustration of crosslinking of hyaluronan with PEGDA using a Michael addition reaction [169].

acids of HA with cysteamine, and then added into

PEGDA (Fig. 9) [169]. However, none of papers
reported the preparation of Dex-based microgel
particles by Michael addition-type reactions to our
best knowledge.

4.4.2. Free radical polymerization

Free radical polymerization of methacrylate-
functionalized Dexs allows for the preparation of
Dex-based hydrogels, as well as nanogels. Various
methacrylated dextrans were synthesized by utiliz-
ing the hydroxy groups of Dex that react with Fig. 10. Chemical structures of various methacrylated dextrans.
different methacrylate precursors. They include
methacrylated Dex derivatives of glycidyl metha- oxy-terminated HEMA-lactate (Dex-LA-HEMA)
crylate (Dex-GMA) [170,171], 2-hydroxyethyl (Fig. 10) [87]. These monomers are homopoly-
methacrylate (dex-HEMA) [172], and hydr- merized or copolymerized with other monomers.
 ARTICLE IN PRESS
460 J.K. Oh et al. / Prog. Polym. Sci. 33 (2008) 448–477
For example, dex-GMA [171] was copolymeri-
zed with amidoethyl methacrylate to prepare
macroporous, cell-adhesive and cell-penetrable
Dex-hydrogels. Dex-LA-HEMA [87] was also co-
polymerized with NIPAM for the preparation of
thermoresponsive hydrogels.
An interesting approach utilized photoinitiated
free radical polymerization in liposomes as reactors
to prepare lipid coated and naked biodegradable
nanogels with tunable degradation properties
[173,174]. In this method, the liposomes were
prepared by multiple extrusions of lipid bilayers
hydrated with dex-HEMA through a polycarbonate
membrane with a well-defined pore size. Photoini-
tiated polymerization was followed in liposomes of
dex-HEMA to form lipid-coated Dex-nanogels with
a diameter of around 400 nm. The surrounding lipids
were removed from lipid-coated nanoparticles by
the addition of a nonionic surfactant, resulting in
the formation of lipid naked nanoparticles [172].
This approach was further extended to prepare
self-rupturing microcapsules, in which nanogels
consisting of copolymers of Dex-HEMA with
dimethylaminoethyl methacrylate (DMAEMA) were
first prepared in liposomes. The resulting nanogels
were then coated with poly(sodium 4-styrenesulfonate)
(PSS) as poly anions and poly(allylamine hydro-
chloride ) (PAH) as polycations using LbL deposi-
tion to form microcapsules consisting of Dex-based
nanogel core with polyelectrolyte shell. The poly-
electrolyte shell was impermeable to dextrans produced
from degradation of nanogel core upon hydrolysis,
leading to rupturing of microcapsules [175,176].
5. Heterogeneous free radical polymerization
Various heterogeneous polymerization reactions
of hydrophilic or water-soluble monomers in the
presence of either difunctional or multifunctional
crosslinkers have been mostly utilized to prepare
well-defined synthetic microgels. They include dis-
persion, precipitation, inverse (mini)emulsion, and
inverse microemulsion polymerization utilizing an
uncontrolled free radical polymerization process.
5.1. Dispersion polymerization
Dispersion polymerization is a technique that
allows for the preparation of micron-sized particles
with narrow size distribution. In the process, most
ingredients including monomers, polymeric stabili-
zers, and initiators are soluble in an organic solvent
as a continuous phase. At the onset, polymerization
occurs in a homogeneous reaction mixture; how-
ever, the formed polymers become insoluble in
the continuous medium, ultimately leading to the
formation of stable dispersion of polymeric particles
with an aid of colloidal stabilizers [177,178]. The
method has been mainly applied to prepare uniform
microspheres of hydrophobic polymers including
polystyrene (PS) [179,180] and poly(methyl metha-
crylate) (PMMA) [181].
Hydrophilic monodisperse micron-sized particles
of PHEMA were also prepared by dispersion
polymerization in the presence of PEO-b-poly
(1,1,2,2-tetrahydroperfluorodecyl acrylate) diblock
copolymer as a stabilizer in supercritical carbon
dioxide [182], and methacryloyl-terminated PMMA
in a 55/45 (wt/wt) mixture of 2-butanol/tolune [183].
Recently, crosslinked microgel particles based on
PHEMA, PGMA, poly(2-hydroxyethyl acrylate)
(PHEA), and poly(2-hydroxypropyl methacrylate)
(PHPMA) were prepared in the presence of difunc-
tional crosslinkers such as ethylene glycol dimetha-
crylate (EGDMA) and 4-[(E)-[(3Z)-3-(4-(acryloyloxy)
benzylidene)-2-hexylidene]methyl]phenyl acrylate.
Drugs and magnetic nanoparticles were either
physically incorporated or chemically attached to
microgels. The resulting microgels were effective as
drug delivery carriers and for DNA applications
[44,184–186].
5.2. Precipitation polymerization
Similar to dispersion polymerization, precipita-
tion polymerization involves the formation of
homogeneous mixture at its initial stage and the
occurrence of initiation and polymerization in
the homogeneous solution. As the formed polymers
are not swellable but soluble in the medium, the use
of crosslinker is necessary to crosslink polymer
chains for the isolation of particles. As a conse-
quence, the resulting crosslinked particles often
have an irregular shape with high polydispersity
(PDI) [178,187–190].
Preparation of hydrogels and microgels based on
PNIPAM and its derivatives by precipitation
polymerization in water has been extensively
explored for biomedical applications [191–193]. This
is due to the thermosensitive properties of PNI-
PAM-microgels that undergo volume change at a
lower critical solution temperature (LCST) in water,
at around 32 1C. Above the LCST, PNIPAM
hydrogels and microgels are hydrophobic and expel
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J.K. Oh et al. / Prog. Polym. Sci. 33 (2008) 448–477
water; below LCST, they are hydrophilic and
swollen in water. These unique properties facilitate
the loading of drugs into microgels as well as
enhance the controllable release of drugs encapsu-
lated in microgels [87]. However, the use of
PNIPAM-based microgels shows a certain limita-
tion to biomedical applications due to relatively
narrow range of physical and chemical properties of
PNIPAM.
An introduction of functional groups is a
promising way to overcome such limitation and
broaden various applications of PNIPAM-based
microgels. Towards that end, various functional
monomers were copolymerized with NIPAM under
precipitation polymerization conditions, resulting in
the preparation of functional microgels [194].
Copolymerization with more hydrophilic or hydro-
phobic monomers enabled to tune the volume phase
transition of the microgels [195,196]. Copolymeriza-
tion with a tetrazole-containing AAm could bind to
various protonated amines in aqueous medium at
millimolar concentration [197]. Dimethylmaleimi-
doethyl acrylamide (DMIAAm) was synthesized
and copolymerized with NIPAM to prepare photo-
crosslinkable poly(NIPAM-co-DMIAAm). These
copolymers aggregated to form particles in water
above of LCST, and in turn the resulting aggregates
were crosslinked by UV irradiation, resulting in the
preparation of microgels [60].
Acid-containing monomers such as AA,
methacrylic acid, and undecanoic acid were copo-
lymerized to prepare PNIPAM-based microgels
with pendent carboxylic acid groups. These car-
boxylic acids provided many advantages. First, they
Fig. 11. Illustration of hydrogel microlens assay, where P(NIPAM-co-AA) microgels were prepared by free-radical precipitation
polymerization method. Reprinted with permission from Ref. [216]. Copyright 2005 American Chemical Society.
461
served as reactive sites for post-modification of
microgels. Examples include the bioconjugation of
P(NIPAM-co-AA) microgels with cell-targeting
biomolecules such as folic acids, proteins, and
antibodies for targeted drug delivery [198–200],
and the reaction of P(NIPAM-co-AA) microgels
with boronic acid derivatives to make a glucose
sensor [201,202]. Second, carboxylic acids in micro-
gels immobilized inorganic precursors through ionic
interactions. Microgels were utilized as polymeric
reactors for semiconductor, metal, and magnetic
nanoparticles [203–205]. In particular, gold-loaded
nanogels were used for site-specific drug delivery
and photodynamic therapy through photothermally
triggered volume transitions under physical condi-
tions [77]. Third, acid-functionalized microgels were
doubly responsive to two external stimuli (tempera-
ture and pH) [206–208]. Fourth, core–shell P
(NIPAM-co-AA) microgels were prepared [209] as
effective delivery carriers of insulin [210], Dox [211],
and anionic surfactant [212]. They were effectively
assembled on an aminopropyltrimethoxysilane-
functionalized glass substrate via electrostatic inter-
actions, resulting in the fabrication of microlens
arrays for biological processes (Fig. 11) [213–216].
Recently, enzyme-responsive hydrogel particles
were prepared by copolymerization of AAm with
an amine-containing PEG-functionalized AAm
in water. The amine groups on microgels were
utilized for attachment of enzyme cleavable peptide
linkers that can be cleaved by a protease
enzyme. The volume change of PAAm-based
microgels upon responsiveness to pH enhanced the
incorporation of drugs and the volume change upon
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462 J.K. Oh et al. / Prog. Polym. Sci. 33 (2008) 448–477
responsiveness to enzymes facilitated the release of
drugs [217,218].
5.3. Inverse (mini)emulsion polymerization
Inverse (mini)emulsion polymerization is a W/O
polymerization process that contains aqueous dro-
plets (including water-soluble monomers) stably
dispersed with the aid of oil-soluble surfactants in
a continuous organic medium. Stable dispersions
are formed by mechanical stirring for inverse
emulsion process and by sonification for inverse
miniemulsion polymerization. Upon addition of
radical initiators, polymerization occurs within the
aqueous droplets producing colloidal particles.
Several reports have demonstrated the synthesis of
hydrophilic or water-soluble particles of PHEMA
[219], PAA [220], and PAAm [219], temperature-
sensitive hollow microspheres of PNIPAM [221],
core–shell nanocapsules with hydrophobic shell and
hydrophilic interior [132], and polyaniline nanopar-
ticles [222]. In addition, this method has also been
utilized to prepare stable organic–inorganic hybrid
particles containing magnetic iron oxide nanoparti-
cles [223,224] and clays [225] in cyclohexane based
inverse miniemulsions. Details on such systems are
also summarized in a review paper [226].
Recently, this method has been explored to
prepare crosslinked microgels in the presence of
difunctional crosslinkers for effective drug delivery.
This is due to a facile confinement of water-soluble
drugs in aqueous droplets dispersed in continuous
Fig. 12. Illustration of preparation for microgels of PEO-b-PPO-b-PEO via inverse emulsion polymerization. Reprinted with permission
from Ref. [229]. Copyright 2005 American Chemical Society.
organic solvents. Temperature- and pH-sensitive
P(NIPAM-co-AA) minigels [227] and PAAm/PAA
interpenetrating polymer network microgels [228]
were prepared in the presence of N,N0 -methylenebi-
sacrylamide. Their swelling properties were studied
in water by measuring particle diameter upon
change in temperature and pH. Stable, crosslinked,
amphiphilic nanoparticles based on acrylated tri-
block copolymer of poly(ethylene glycol)-b-poly
(propylene glycol)-b-poly(ethylene glycol) (PEO-b-
PPO-b-PEO) were prepared by inverse emulsion
photopolymerization (Fig. 12). The hydrophobic
PPO-rich domains enhanced incorporation of Dox,
an amphiphilic anticancer drug, up to 9.8 wt%. The
resulting microgels had the diameter of 50 and
500 nm [229]. In addition, poly(magnesium acrylate)
microgels with the diameter of 40 mm were prepared
for immobilizing enzymes such as Glucose oxidase
(Gox) as bioreactors [230].
Biodegradable microgels were prepared via poly-
merization of a macromonomer of diacrylated
Pluronic–oligo(lactic acid) copolymer, which was
synthesized by a ring opening polymerization of
D,L-lactide initiated with PEO-b-PPO-b-PEO, fol-
lowed by reaction with acryloyl chloride [231]. The
size of the resulting microgels varied from 200 to
600 mm as a stirring speed decreased during poly-
merization; however their size distributions were
relatively monodisperse. BSA as a model protein
was encapsulated and its release behavior was
examined as a function of temperature [232]. Acid-
degradable microgels based on PAAm were also
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J.K. Oh et al. / Prog. Polym. Sci. 33 (2008) 448–477 463

prepared for delivery of protein and pDNA
[233,234]. A water-soluble diacrylamide crosslinker
containing an acid-labile acetal group was synthe-
sized [93] and introduced into inverse emulsion poly-
merization. The resulting microgels were degraded
under acidic condition (pH ¼ 5.0) to the correspond-
ing linear polymers, resulting in the release of
encapsulated molecules. In addition, acid-degradable
cationic microgels were prepared by introducing
2-acryloxyethyltrimethylammonium chloride (AET-
MAC), a cationic monomer, for antigen delivery.
5.4. Inverse microemulsion polymerization
While inverse (mini)emulsion polymerization
forms kinetically stable macroemulsions at, below,
or around the critical micellar concentration
(CMC), inverse microemulsion polymerization pro-
duces thermodynamically stable microemulsions
upon further addition of emulsifier above the
critical threshold. This process also involves aqu-
eous droplets, stably dispersed with the aid of a
large amount of oil-soluble surfactants in a con-
tinuous organic medium; polymerization occurs
within the aqueous droplets, producing stable
hydrophilic and water-soluble colloidal nanoparti-
cles having a diameter of less than 50–100 nm
[235,236]. This method has been explored for the
preparation of well-defined nanoparticles based on
PNIPAM [237,238], PAAm [239–241], and poly
(dimethylacrylamide) [242] as well as magnetic
polymeric particles of PAAm and PEO [75] contain-
ing iron oxide nanoparticles [243].
Inverse microemulsion polymerization was ex-
plored for the synthesis of well-defined nanogels.
Poly(vinylpyrrolidone)-based nanogels incorporated
with Dex as a water-soluble macromolecular
carbohydrate drug were prepared [244,245]. Catio-
nic nanogels of poly(HEA-co-AETMAC) were
prepared in the presence of oligo(ethylene glycol)
dimethacrylate (OEGDMA) as a crosslinker. The
diameter of the resulting nanogels decreased from
150 to 40 nm as the amount of crosslinker increased.
The resulting nanogels had potential for gene
delivery, since the presence of quaternary ammo-
nium ion side groups appeared to enhance incor-
poration of DNA into nanogels via electrostatic
association with the phosphate groups [246]. Poly
(amino acid)-based nanoparticles with different
surface PEGylation were prepared. a,b-Poly
(N-2-hydroxyethyl)-D,L-aspartamide (PHEAS) and
PEG-modified PHEA (PHEAS-PEG) were functio-
nalized with a methacrylate group and then poly-
merized by UV irradiation in inverse microemulsion.

Fig. 13. Fluorescence images of rat C6 glioma cells after incubation with free meta-tetra(hydroxyphenyl)chlorine as a photosensitizer
(upper) and ultrafine hydrophilic PAAm-based nanogels incorporated with meta-tetra(hydroxyphenyl)chlorine (lower): (a, d) before light
exposure, (b, e) after light exposure, and (c, d) zoom out of (b) and (e). Scale bar ¼ 30 mm for (a), (b), (d), and (e), and 40 mm for (c) and (f).
Reprinted with permission from Ref. [248]. Copyright 2006 Wiley InterScience.
 ARTICLE IN PRESS
464 J.K. Oh et al. / Prog. Polym. Sci. 33 (2008) 448–477
The resulting nanoparticles had a size of around
250 nm in diameter by TEM. The fluorescein-loaded
PHEA-based nanoparticles were prepared in the
presence of fluorescein sodium salts, and examined
for cellular uptake using macrophage cells [247].
Recently, ultrafine hydrophilic PAAm-based na-
nogels incorporated with meta-tetra(hydroxyphe-
nyl)chlorine (mTHPC) as a photosensitizer were
prepared for photodynamic therapy. In the process,
both AAm and N,N-methylene(bis acrylamide) as a
crosslinker were directly emulsified into hexane/
Aerosol OT, without water, which allowed for the
preparation of tiny PAAm nanoparticles with a
diameter of 2–3 nm [248]. The presence of water
in hexane/Aerosol OT produced 20 nm diameter
PAAm particles with a relatively broad size
distribution [249]. The resulting PAAm-nanogels
incorporated with mTHPC were effective to kill rat
C6 glioma cells upon exposure to 650 nm visible
light (Fig. 13).
6. Heterogeneous controlled/living radical
polymerization
CRP provides a versatile route for preparation of
(co)polymers with controlled molecular weight,
narrow molecular weight distribution (i.e., Mw/Mn,
or PDIo1.5), designed architectures, and useful
end-functionalites [250–252]. Recently, CRP has
been explored as a tool to preparation of well-
controlled polymer–protein/peptide bioconjugates
[253]. Various methods for CRP have been devel-
oped; however, the most successful techniques
include atom transfer radical polymerization
Fig. 14. Preparation of stable nanoparticles of well-controlled poly(oligo (ethylene glycol) monomethyl ether methacrylate) with
Mw/Mno1.3. Evolution of molecular weight and molecular weight distribution (a) and CONTIN plot of particles dispersed in cyclohexane
inverse miniemulsion (b). Inset is their atomic force microscopy (AFM) image. Reprinted with permission from Ref. [297]. Copyright 2006
American Chemical Society.
(ATRP) [254–260], stable free radical polymeriza-
tion (SFRP) [261,262], and reversible addition-
fragmentation chain transfer (RAFT) polymeriza-
tion [263–265]. Peptide sequences [266,267], biotin
[268], and streptavidin [269] have been successfully
modified to become ATRP initiators. Many CRP
reactions of hydrophobic monomers have been
examined in heterogeneous aqueous dispersions,
including conventional emulsion, microemulsion,
miniemulsion, dispersion, and suspension media.
They resulted in the preparation of stable latex
particles consisting of well-defined hydrophobic
polymers. Several reviews [270–273] and papers
concerning CRP in aqueous dispersion have been
published using ATRP [274–281], SFRP [282–288],
and RAFT [289–295].
However, only a few reports on CRP of water-
soluble or hydrophilic monomers in heterogeneous
media have been published. The first successful
CRP using ATRP in inverse miniemulsion described
the synthesis of stable nanoparticles of well-con-
trolled water-soluble poly(oligo(ethylene glycol)
monomethyl ether methacrylate) (POEOMA) with
Mw/Mno1.3. The resulting nanoparticles had uni-
form size distribution (Fig. 14) [296,297]. This
method has also been applied for the synthesis of
stable nanoparticles of well-controlled hydrophilic
PEO-b-PHEMA block copolymers with Mw/Mno
1.3. Interestingly, the resulting doubly hydrophilic
block copolymers self-assembled to form well-
defined micelles with a diameter of 10–20 nm in
water [298]. In addition, a RAFT inverse miniemul-
sion polymerization was also reported for the
preparation of hydrophilic latex of PAAm [299].
 ARTICLE IN PRESS
J.K. Oh et al. / Prog. Polym. Sci. 33 (2008) 448–477 465

CRP techniques have been explored for the bioconjugated with fluorescein isothiocyanate
synthesis of gels [300–305] and crosslinked nano- (FITC)-labeled avidin. The number of biotin
particles of well-controlled polymers in the presence molecules in each nanogel was determined to be
of crosslinkers. Hydrogel nanoparticles of PNIPAM 142,000 and the formation of bioconjugates of
was prepared by precipitation polymerization via nanogels with avidin was confirmed using fluores-
ATRP in water [306]. Acid cleavable poly(butyl cence microscopy (Fig. 16) [308].
acrylate) microgel core surrounded with linear Second, they enable incorporation of a high
POEOMA shell for delivery of hydrophobic drugs loading level of drugs such as the anticancer
were prepared by combination of acid-labile micro- drug Dox after polymerization [308] and water-
gel approach with RAFT-mediated seeded disper- soluble biomacromolecules such as rhodamine B
sion polymerization [307].
ATRP in inverse miniemulsion for the synthesis
and functionalization of stable (bio)degradable
crosslinked nanogels of well-controlled water-solu-
ble polymers in the presence of a disulfide-functio-
nalized dimethacrylate (DMA) was reported
[297,308]. This approach allowed for the prepara-
tion of biomaterials with many useful features
(Fig. 15).
First, the resulting particles preserve a high
degree of halide end-functionality to enable further
chain extension to form functional block copoly-
mers and facile functionalization with biorelated
molecules, such as by utilizing click reactions
[309–311]. Further, several other ways are available
for the preparation of novel functional nanogels
using the method involving ATRP in inverse
miniemulsion. They include the use of functional
ATRP initiators and the facile copolymerization
with functional monomers. The latter approach
was successfully demonstrated with the preparation
Fig. 16. Fluorescence microscopy image of FITC–avidin–
of OH-functionalized nanogels by copolymerizing biotin–nanogel conjugates. Scale bar ¼ 5 mm. Reprinted with
with 2-hydroxyethyl acrylate (HEA). The nanogels permission from Ref. [308]. Copyright 2006 American Chemical
were then conjugated with biotin, and further Society.

Fig. 15. Illustration of unique features of stable biodegradable nanogels of well-controlled water-soluble polymers in the presence of a
disulfide-functionalized dimethacrylate for effective targeted drug delivery.
 ARTICLE IN PRESS
466 J.K. Oh et al. / Prog. Polym. Sci. 33 (2008) 448–477
isothiocyanate (RITC)-labeled Dex during polymer-
ization [312]. Furthermore, the RITC-dextran-
loaded nanogels could enter cells through an
endocytosis. Fig. 17 shows laser confocal fluores-
cence microscope image of RITC-Dex loaded
nanogels (green) within MC3TC cells stained for
nucleus (blue) and actin (red).
Third, they are degradable in a reducing en-
vironment to individual polymeric chains with a
relatively narrow molecular weight distribution
Fig. 17. Laser confocal fluorescence microscopy image of green
RITC-Dex loaded nanogels (abs. 570 nm (red), but the color was
changed to green to distinguish it from the actin), blue nucleus
(abs. 350 nm, Hoechst blue) and red actin (abs. 649 nm cy5-
conjugated phalloidin), demonstrating endocytosis of the nanogels.
Fig. 18. Nanogels containing RITC-labeled dextran in water before and after the addition of dithiothreitol (DTT) (a); and fluorescence
microscopy images of nanogels loaded with RITC-dextran before (b) and after (c) degradation in water. The scale bars in b and c are
50 mm. Reprinted with permission from Ref. [312]. Copyright 2007 American Chemical Society.
(Mw/Mno1.5), indicating the formation of a uni-
formly crosslinked network in the individual
particles. This uniform structure is anticipated to
improve control over the release of encapsulated
agents (Fig. 18). More importantly, nanogels are
biodegraded in the presence of a biocompatible
glutathione tripeptide, which is commonly found in
cells at mM concentrations [92,313–315]. The
biodegradation of nanogels can trigger the release
of encapsulated molecules as well as facilitate the
removal of empty vehicles.
Fourth, properties including swelling ratio, de-
gradation behavior, and colloidal stability of
nanogels prepared by ATRP are superior to those
prepared by conventional free radical inverse
miniemulsion polymerization. Fifth, nanogels will
enhance circulation time in the blood, since they
consist of POEOMA, an analog of linear PEO,
which can prevent nanoparticle uptake by reticu-
loendothelial system (RES) [80].
Well-defined functional nanogels prepared by this
newly developed method hold great potential as
drug delivery carriers to target-specific cells for
biomedical applications. Future nanogels will con-
tain targeting residues such as peptides, proteins,
and antibodies for delivery to specific regions and
cells.
7. Conclusion
Recent developments of microgel/nanogel parti-
cles as drug delivery carriers for biological and
biomedical applications were reviewed in this paper.
Major synthetic strategies for the preparation of
microgels/nanogels were described in detail. Photo-
 ARTICLE IN PRESS
J.K. Oh et al. / Prog. Polym. Sci. 33 (2008) 448–477
lithographic methods have been explored for pre-
paration of well-defined 3D hydrogel particles and
microgel rings. In particular, the PRINT method
allowed for the preparation of monodisperse sub-
micron-sized microgels with control over particle
size and composition, and various shapes including
trapezoidal, bar, conical, and arrow. However, the
methods require the development of surface treat-
ment of stamps or new materials for replica molds
to enhance a facile release of a molded gel from
stamps or replica molds. Micromolding methods of
hydrogels allowed for fabrication of submicron-
sized microgels. Furthermore, they can minimize the
need for the use of costly lithographic equipment
and clean room facilities. Continuous microfluidic
preparation of microgels involves two steps: emul-
sification of monomers by breaking up the liquid
threads to droplets and in-site crosslinking of the
droplets by photopolymerization or polycondensa-
tion. Chemical gelation and physical gelation by
temperature change, reversible shear thinning, and
ionic crosslinking, and coalescence-induced gelation
methods have been developed. In particular, ionic
crosslinking using external gelation resulted in
microfluidic preparation of stable monodisperse
spherical alginate microgels, while internal cross-
linking led to weak gelation with poor control over
microgel morphology. Modification of naturally
occurring carbohydrate-based biopolymers such as
CS, HA, and Dex has been explored for the
preparation of functional microgels for targeted
drug delivery. This is due to unique properties of
these biopolymers, including non-toxicity, high
water solubility, biocompatibility, biodegradability
as well as high degree of functionality. Inverse
(mini)emulsion, reverse micelle, membrane emulsi-
fication, aqueous homogeneous gelation, spray
drying, Michael addition, and free radical polymer-
ization have been developed.
The most general methods for the preparation of
well-defined synthetic microgels are heterogeneous
polymerization of hydrophilic or water-soluble
monomers in the presence of either difunctional or
multifunctional crosslinkers. Free radical disper-
sion, precipitation, inverse (mini)emulsion, and
inverse microemulsion polymerization have been
explored. Dispersion polymerization generally pro-
duced micron-sized particles. Precipitation polymer-
ization has been extensively explored to prepare
microgels based on PNIPAM and its derivatives,
due to their volume change at LCST. Copolyme-
rization with functional monomers broadened
467
various applications of PNIPAM-based microgels.
Inverse (mini)emulsion polymerization utilizes a
W/O dispersion that provides a facile confinement
of water-soluble drugs in aqueous droplets dis-
persed in continuous organic solvents. Stable
microgels based on amphiphilic PEO-b-PPO-b-
PEO and Pluronic–oligo(lactic acid) copolymer,
and acid-degradable microgels based on PAAm
were developed for effective delivery of drugs,
proteins, and DNA. Inverse microemulsion poly-
merization involves thermodynamically stable
microemulsions, allowing for the preparation of
well-defined nanogels. Poly(vinylpyrrolidone)-based
nanogels, cationic nanogels of poly(HEA-co-AET-
MAC), and poly(amino acid)-based nanogels were
prepared. In particular, ultrafine PAAm-based
nanogels incorporated with a photosensitizer were
developed for photodynamic therapy.
CRP techniques have been recently explored to
prepare stable microgels/nanogels of well-controlled
polymers. A new method utilizing ATRP, inverse
miniemulsion polymerization, and thiol-disulfide
exchange reaction allowed for the development
and functionalization of stable biodegradable
nanogels of well-controlled water-soluble polymers
in the presence of a disulfide-functionalized di-
methacrylate (DMA). This approach allowed for
the preparation of biomaterials with many useful
features; preservation of a high degree of halide end-
functionality for facile functional block copolymer-
ization and bioconjugation, biodegradation in the
presence of a biocompatible glutathione tripeptide,
formation of uniform networks, larger and better
swelling ratio, degradation behavior, and colloidal
stability compared to conventional counterparts,
high loading level of anticancer drugs, and en-
hanced circulation time in the blood.
Future design and development of effective micro-
gel-based DDSs for in vivo applications require a high
degree of control over properties. These include
excellent stability for prolonged circulation in the
blood stream, novel functionality for further biocon-
jugation; dimension less than 200 nm in diameter, and
biodegradability for facile removal of the empty
device and sustained release of drugs.
One future goal of research in this area should be
the improved design of microgels/nanogels with
specific targeting residues to enable highly selective
uptake into particular cells. This will be especially
important for the targeting of cancer cells, thereby
reducing non-specific uptake into healthy cells.
Furthermore, it will increase the efficiency of the
 ARTICLE IN PRESS
468 J.K. Oh et al. / Prog. Polym. Sci. 33 (2008) 448–477
DDS. As the bridge between chemistry and biology
continues to become shorter, biologists will need to
elucidate the specific interactions between biomole-
cules and cellular integral receptors. Chemists can
then design microgels/nanogels to present these
proteins or antibodies on the surface of the
microgels/nanogels as a way to precisely target-
specific cells. In this way, careful control over
stability, size, biodegradability, and functionality
for bioconjugation will guide the development of
the next generation of microgels/nanogels.
Acknowledgments
The Dow Chemical Co. is greatly acknowledged
for the support of the work and the permission to
publish this work. J.K.O thanks the NSERC
Postdoctoral Fellowship of Canada for financial
support during his stay at Carnegie Mellon Uni-
versity.
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