Advanced Drug Delivery Reviews 61 (2009) 768–784

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Advanced Drug Delivery Reviews

j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / a d d r

Intelligent polymeric micelles from functional poly(ethylene glycol)-poly(amino acid)

block copolymers☆
Younsoo Bae a, Kazunori Kataoka b,c,d,⁎
a
Department of Pharmaceutical Sciences, College of Pharmacy, University of Kentucky, 725 Rose Street, Lexington, KY 40536, USA
b
Department of Materials Engineering, Graduate School of Engineering, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8656, Japan
c
Center for Disease Biology and Integrative Medicine, Graduate School of Medicine, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan
d
Center for NanoBio Integration, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8656, Japan

a r t i c l e i n f o a b s t r a c t

Article history: This review describes our recent efforts on the design and preparation of intelligent polymeric micelles from
Received 14 September 2008 functional poly(ethylene glycol)-poly(amino acid) (PEG-PAA) block copolymers. The polymeric micelles
Accepted 29 April 2009 feature a spherical sub-100 nm core–shell structure in which anticancer drugs are loaded avoiding
Available online 5 May 2009
undesirable interactions in vivo. Chemical modification of the core-forming block of PEG-PAA with a
hydrazone linkage allows the polymeric micelles to release drugs selectively at acidic pH (4–6). Installation
Keywords:
of folic acids on the micelle surface improves cancer cell-specific drug delivery efficiency along with pH-
Intelligent polymeric micelles
PEG-poly(amino acid) block copolymers
controlled drug release. These intelligent micelles appear to be superior over classical micelles that physically
Controlled drug delivery incorporate drugs. Studies showed both controlled drug release and targeted delivery features of the micelles
reduced toxicity and improved efficacy significantly. Further developments potentiate combination delivery
of multiple drugs using mixed micelles. Therefore clinically relevant performance of the polymeric micelles
provides a promising approach for more efficient and patient-friendly cancer therapy.
© 2009 Elsevier B.V. All rights reserved.

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 769
2. Classical polymeric micelles with physical drug entrapment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 769
2.1. Poly(ethylene glycol)-poly(amino acid) amphiphilic . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 769
2.2. Micelle formation and physicochemical properties . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 771
3. pH-sensitive polymeric micelles with covalent drug conjugation. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 772
3.1. Drug conjugation via pH-labile hydrazone linkage . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 772
3.2. Tissue penetration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 775
4. Folate-conjugated polymeric micelles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 776
4.1. Folic acid and active cancer targeting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 776
4.2. Cellular interaction and bioactivity response . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 777
5. Antitumor activity and bioavailability . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 778
5.1. In vitro cytotoxicity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 778
5.2. Biodistribution . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 778
5.3. In vivo antitumor activity. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 779
6. Combination chemotherapy using polymeric micelles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 780
6.1. Modification of delivery environment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 780
6.2. Concurrent delivery of multiple therapeutic agents. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 781
7. Future prospects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 782
Acknowledgment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 782
References. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 782

☆ This review is part of the Advanced Drug Delivery Reviews theme issue on “The Role of Gene- and Drug Delivery in Women's Health — Unmet Clinical Needs and Future
Opportunities”.
⁎ Corresponding author. Department of Materials Engineering, Graduate School of Engineering, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8656, Japan. Tel.: +81
3 5841 7138; fax: +81 3 5841 7139.
E-mail address: kataoka@bmw.t.u-tokyo.ac.jp (K. Kataoka).

0169-409X/$ – see front matter © 2009 Elsevier B.V. All rights reserved.
doi:10.1016/j.addr.2009.04.016
 Y. Bae, K. Kataoka / Advanced Drug Delivery Reviews 61 (2009) 768–784
1. Introduction
Molecular targeted therapies, taking advantages of differential
metabolism and expression levels of certain proteins in lesions, are
generally considered the most efficient way to treat diseases [1]. In
theory the targeted therapies can achieve the maximum therapy with
the minimum side effects. In reality, delivering potent drugs to where
they are necessary is still challenging for clinicians and pharmaceu-
tical scientists. It is because most of potent drugs are small molecules
that can freely diffuse in both benign and malignant diseased cells.
This induces non-specific drug distribution in the body. Even if the
drugs are highly specific to certain molecular targets, they often suffer
from low solubility, rapid metabolism or antagonistic interaction with
other drugs. Therefore, it is almost impossible to simultaneously
control the bioactivities and physicochemical properties of multiple
drugs, overcoming aforementioned pharmacokinetic barriers.
Drug delivery systems (DDS) appeared to be a promising and
reliable approach to deliver potent drugs to the site of action precisely
and timely. Preclinical and clinical studies constantly showed that DDS
using natural and artificial macromolecules resulted in successful
cancer therapy with reduced toxicity and improved efficacy [2]. A
mechanism for tumor-specific delivery is explained by characteristic
tumor microenvironments. Tumor tissues are characterized with
leaky blood vessels and the premature lymphatic drainage. Due to the
high molecular weight and large hydrodynamic radii, macromolecules
circulate in the blood for a longer period of time than small molecules.
These macromolecules were observed to preferentially accumulate in
the tumor tissue, and retain within the tissues for a prolonged period
of time. This phenomenon is referred to as the enhanced permeability
and retention (EPR) effects, providing a key rational of DDS using
macromolecular drug carriers [3].
Currently available macromolecular drug carriers may include
water-soluble polymers, dendrimers, polymeric micelles, and lipo-
somes [4–7]. Each carrier has advantageous features to provide struc-
tural flexibility, multiple functional moieties, a sequestered nano
depot, and robust stability, respectively. Among these carriers, only
the polymeric micelles undergo dynamic physicochemical changes
during drug entrapment and release in terms of molecular assembly
and dissociation between block copolymer components. The poly-
meric micelles are spherical supramolecular nanoassemblies prepared
from self-assembling amphiphilic block copolymers (Fig. 1). They
feature a sub-100 nm core–shell structure, which provides a nano
depot for hydrophobic drugs enveloped with a hydrophilic shell,
improving drug solubility [8]. The hydrophilic shell suppressing
protein adsorption allows the polymeric micelles to avoid foreign
body reaction while improving drug solubility. This property is called
stealth functionality. Because of their characteristic structure and
stealth functionality, the polymeric micelles can stably transport
bioactive molecules to the tumor tissues suppressing the immune
response and non-specific drug distribution to the normal tissues.
In this review, the polymeric micelles are categorized into two
groups depending on drug-loading methods. One group is for classical
‘physical drug entrapment type micelles’ and the other one is for
2nd generation ‘covalent drug conjugation type micelle’. As for the
Fig. 1. Amphiphilic block copolymers and polymeric micelles.
769
physical drug entrapment type micelles, they incorporate drug
payloads through the hydrophobic interaction in the micelle core
[9]. Drugs can be entrapped also in gel-like amorphous core. In either
case, the equilibrium rates determine the physicochemical stability
and drug release patterns of the polymeric micelles, which are
controlled time-dependently. In contrast, covalent drug conjugation
type micelles have drug-binding linkers that stably tether drugs in the
micelle core until the polymeric micelles accumulate in the site of
action and are exposed to the in vivo stimuli such as ions, endogenous
signal peptides, enzymes, and pH that trigger drug release [10]. Drug
conjugation through metal chelate as well as environment-sensitive
drug conjugation might be included this category [11,12]. Covalent
drug conjugation type micelles appear to be more stable than physical
drug entrapment type micelles as long as the linkage remains intact.
Since their drug release patterns can be modified according to
chemical stability of drug-binding linkers, covalent drug conjugation
type micelles provide environment-responsive controlled drug
release systems, or also called intelligent systems [13].
If the core of the micelles is modified for drug entrapment, the
surface of the micelles can be functionalized with targeting molecules
that can specifically interact with certain molecular targets on the
lesions [14]. Receptors, intracellular organelles, and signal peptides
are such examples. Antitumor activity and bioavailability of polymeric
micelles can be improved further by installing targeting molecules
that are specific to malignant cell membrane transporters or intra-
cellular proteins [15]. Targeting tissues using drug carriers is known as
‘active targeting’ technology. Both physical drug entrapment and drug
conjugation type micelles can be used for active targeting. In com-
parison to active targeting, the method to deliver drugs using carriers
to the tumor tissues solely through the EPR effects is called ‘passive
targeting’. Receptor recognition and its accompanying cellular inter-
action and response are crucial issues on the design of effective drug
carriers. Indeed cellular response and drug efficacy increase signifi-
cantly by simply changing drug delivery approaches because targeting
molecules can enhance interactions between the polymeric micelles
and the cells [16]. This widens the therapeutic window of drug
payloads, eventually improving their bioavailability.
Characteristic properties of the polymeric micelles can be derived
from the design of amphiphilic block copolymers. In vivo behaviors
and the long-term fate of the polymeric micelles are fundamentally
influenced by the properties of the block copolymers. Most impor-
tantly, therapeutic efficacy is determined by both drug delivery and
release strategies for the micelles based on the pharmacokinetic (PK)
profiles. Polymeric micelles with optimized design and compositions,
therefore, can properly protect, deliver, and release multiple potent
drugs to the targeted site with an identical PK profile. This new class of
approach in drug delivery represents a promising methodology for
drug combination.
In order to get a better understanding of aforementioned proper-
ties, the design, preparation and performance of the polymeric
micelles are extensively summarized in this review, based mainly on
our previous reports. Particular attention was paid to the correlation
between carrier design and cancer treatment efficacy, demonstrating
significance of polymeric micelles on cancer chemotherapy. Lastly, it
must be emphasized that the approach and methodology described
herein will also likely be exploited to develop the carriers for other
bioactives such as imaging agents, peptides, functional nucleic acids,
and plasmids [17].
2. Classical polymeric micelles with physical drug entrapment
2.1. Poly(ethylene glycol)-poly(amino acid) amphiphilic
block copolymers
Amphiphilic block copolymers consist of multiple segments with
distinct solubility against certain solvents [18]. Generally two or three
770 Y. Bae, K. Kataoka / Advanced Drug Delivery Reviews 61 (2009) 768–784
segments are conjugated linearly to prepare amphiphilic block
copolymers. Depending on the thermodynamic conditions, amphi-
philic block copolymers may form nano structures such as lamellas,
globules, cylinders, vesicles, and micelles [19]. In particular amphi-
philic block copolymers from hydrophilic and hydrophobic segments
undergo spontaneous self-assembling in the aqueous solutions,
sequestering hydrophobic segments from aqueous environments by
hydrophilic segments [20,21]. This phenomenon is useful to dissolve
hydrophobic and fatty materials as likely seen in low molecular
weight surfactants (LMWS) forming micelles. Compared to LMWS
micelles, polymeric micelles prepared from amphiphilic block
copolymers are superior in stability and most noticeably in high
capacity for the incorporation of guest molecules in the core.
Although any types of amphiphilic block copolymers could form
micelle structure, extensive studies have shown that poly(ethylene
glycol)-poly(amino acids) (PEG-PAA) block copolymers are possibly
the most facile components to design the polymeric micelles [22]. PEG
is a biocompatible water-soluble polymer, which is widely used in the
biopharmaceutical industry because of its excellent water solubility,
chain mobility, and non-immunogenicity. PEG is non-toxic and can be
cleared from the body through renal filtration with a molecular weight
of less than 30,000 [23]. When it comes to PAA, it is also highly
biocompatible, non-toxic, and economic with versatile functional
groups such as hydroxyl, carboxyl, amino, and thiol groups. Such a
variety of functional groups is of great benefit to modify the chemical
structure of the core for drug conjugation.
Amino acids can be polymerized by traditional solid-phase peptide
synthesis methods. A large number of oligomers and short peptides
can be easily synthesized by this well-established method. One of the
most common synthetic routes for polypeptide synthesis is poly-
condensation of monomers protected at one carboxyl and activated
for polymerization at the other, followed by protecting group removal.
A set of recursive protection/conjugation/deprotection process allows
one to construct artificial PAA chains with theoretically unlimited
sequences. It is generally accepted that this procedure gives rise to
oligomer polypeptides. When synthesizing homopolymers with chain
length longer than 10 repeating units, activated amino acid monomers
are used for the polymerization. This method is more facile and
Fig. 2. Amino acid N-carboxy anhydride synthesis and poly(amino acid) polymerization.
convenient in terms of saving time and efforts for preparation as well
as purification of PAA, accompanied with no repetitive protection/
deprotection process.
Activated amino acid monomers can be in form of activated esters
or N-carboxy anhydride (NCA). NCA synthesis methods may include
Leuchs, Curtius and Fuchs–Farthing methods. Among these methods,
the Fuchs–Farthing method is favorably used because pure NCA with a
good yield can be easily obtained. Briefly, amino acids are reacted in
inert polar solvents such as ethylacetate, dioxane, THF, and acetoni-
trile, in the presence of phosgene (Fig. 2). Once NCA is obtained,
polymerization is initiated using primary amino compounds. For PEG-
PAA block copolymer synthesis, α-methoxy ω-amino PEG is generally
used as a macro initiator. Hetero bifunctional PEG can be also used for
the reaction, yet in this case, end group other than amino group must
be protected. PEG with molecular weight between 2 K and 20 K has
been confirmed previously to initiate polymerization of NCA success-
fully [24].
In early studies, poly(ethylene glycol)-poly(β-benzyl L-aspartate)
(PEG-PBLA) block copolymers are synthesized to prepare the poly-
meric micelles [25,26]. Selection of the solvent is of crucial importance
because polypeptide chains in the solutions can perturb amino group
activity at the chain end during the polymerization. For instance, in
PEG-PBLA synthesis, α-helix, β-sheet, and random coil conformation
of poly(aspartic acids) [P(Asp)] often resulted in different quality of
product in terms of polydispersity and molecular weight [27–29].
These formations influence the mobility of active amino end group
within the chains, which often results in a broad polymer distribu-
tion in a high degree of polymerization. Accumulating data show that
P(Asp) with the numbers of repeating units between 10 and 30, 30
and 50, and over 50 can be successfully synthesized in dimethylfor-
mamide, dimethylsulfoxide, and dichloromethane, respectively.
The balance between PEG and PBLA segments is of importance
to prepare stable polymeric micelles for certain therapeutic agents
because the ratio between hydrophilic and hydrophobic segments
directly influences self-assembly structures and stability. Side chains of
P(Asp) are activated or chemically modified further, introducing func-
tional groups or conjugating drugs. For these reasons, block copolymer
synthesis using other amino acid NCAs has been extensively studied.
 Y. Bae, K. Kataoka / Advanced Drug Delivery Reviews 61 (2009) 768–784
Aspartic acid, glutamic acid, and lysine appeared to be the most
appropriate amino acids for NCA synthesis, in terms of the synthesis,
recrystallization, and polymerization [30]. These PEG-PAA block
copolymers can be used for preparing polymeric micelles for various
drugs with various functional groups.
2.2. Micelle formation and physicochemical properties
Micelle formation of amphiphilic block copolymers is accompa-
nied with minimizing free energy, and entropy change is generally
considered the most important factor to form stable polymeric
micelles [31]. When it comes to preparing drug-loaded polymeric
micelles, influence of enthalpy becomes also important to micelle
formation. Our early studies have shown the importance of providing
the most thermodynamically favorable conditions on micelle forma-
tion, adjusting concentrations, temperature and solvents along with
block copolymer compositions.
During the process of the entropy-driven micelle formation, the
concentrations of polymers in solutions would be the most important
factor [32]. Indeed a critical micelle concentration, or CMC, varies
depending on block copolymer types and compositions. It generally
ranges between 10− 6 and 10− 7 M, which is 1000 times lower than
that of LMWS micelles [33]. Interestingly, although it is obvious that
the polymeric micelles are stable above the CMC, chain exchange can
occur irrespective of the CMC level [34,35]. This intriguing phenom-
enon demonstrates that polymeric micelle formation is thermodyna-
mically stabilized yet not completely frozen. Nevertheless it must be
noted that the kinetic of chain exchange between the polymeric
micelles is extremely slow compared to LMWS micelles. Also, the
chain exchange between the polymeric micelles is suppressed as the
micelle core becomes hydrophobic by drug entrapment. It maintains
the polymeric micelles stable until they are used for the treatments.
Polymeric micelles can be prepared mainly by two methods. The
most general method is through dialysis. Technically amphiphilic
block copolymers are dissolved first in organic solvents homoge-
neously. These organic solvents must be miscible well with aqueous
solutions. Homogeneous polymer solutions are then diluted roughly
using aqueous solutions, followed by dialysis until organic solvent is
removed completely. The micelle solutions can be either freeze-dried
or concentrated further through ultrafiltration and rotatory evapora-
tion for the future use. Second method is combination of emulsifica-
tion, evaporation, and sonication. Block copolymers are dissolved in
organic solvents such as methanol, acetonitrile, and dichloromethane
in a round-bottom flask. Solvents are then evaporated to form a thin
film or gel. Aqueous solutions are added to the flask at the same
temperature and the mixed solutions are sonicated to form polymeric
micelles. The solution is evaporated further to remove organic solvent
completely. Organic solvent content can be determined by gas
chromatography and must be controlled to be relevant to clinical
applications. Obtained micelle solutions can be concentrated further
as described above. This method is relatively convenient yet polymers
that form crystals on the bottom of the flask, which cannot be
reconstituted easily in the aqueous solutions, may not give a good
result. Another possible method to prepare micelles is solution spray
process, which generally induces co-precipitation of reactants and
reactors above critical micelle concentration. However, this method
requires special equipments and the reaction conditions should be
optimized. Therefore, it may not be appropriate for laboratory scale
micelle preparation.
Physical drug entrapment is generally carried out during the
process of micelle formation. Amphiphilic block copolymers may be
dissolved in aqueous solutions directly to form micelles, yet particle
size would become larger with a broad distribution, compared to the
polymeric micelles prepared through the dialysis method. This
reduces drug-loading efficiency. Drug entrapment experiments to
the polymeric micelles have been extensively conducted using
771
adriamycin (ADR) and PEG-PBLA based polymeric micelles [34].
ADR, which is a product name of doxorubicin (DOX), is effective
against leukemia, breast carcinoma and other solid tumors. Yet its
clinical application has been limited owing mainly to side effects such
as cardiotoxicity, myelosuppression, and nephrotoxicity. As a model
drug, ADR has relevant hydrophobicity, multiple functional groups
modifiable, and UV absorption with fluorescence emission. This is the
reason why ADR is widely used in many studies.
In early studies, ADR molecules were conjugated to natural
macromolecules like transferrin or to artificial hydrophilic polymers.
Although these systems reduced drug toxicity, most of them often
suffered from reduced bioactivity of conjugated drugs. In this regard,
the polymeric micelles exemplified a new approach to resolve these
issues [36]. Later studies showed that amphiphilic block copolymers
comprising of PEG-PBLA formed a core–shell micelle structure. ADR
was then incorporated in PEG-PBLA polymeric micelles via physical
entrapment. Although PEG-PBLA/ADR physical entrapment type
micelles showed a potential of polymeric micelles as a drug carrier,
drug entrapment and micelle stability were low due to poor affinity of
PBLA segment with ADR. PBLA did not provide enough affinity to
entrap ADR molecules in the core stably. In order to improve drug
entrapment efficiency, PEG-PBLA was chemically modified by con-
jugating ADR directly to the block copolymers [37,38]. Prepared PEG-
poly(aspartate-adriamycin) [PEG-p(Asp-ADR)] block copolymers
showed a better drug entrapment efficiency for ADR possibly due
to the π–π interaction between drug molecules (Fig. 3). ADR was
conjugated to the side chain of the P(Asp) segment of the block
copolymer between the carboxylic groups of the P(Asp) segment and
its glycosidic primary amino group of ADR. Approximately 50% of the
carboxylic moieties in the P(Asp) segment were conjugated with ADR,
making the P(Asp) segment hydrophobic enough to form micelles in
aqueous solutions. The high polymer content causes coagulation or
gelation in most drug–polymer conjugates but no such phenomena
were seen in these micelles. To the contrary, the self-association
between ADR molecules through the π–π interaction increased the
cohesive force within the core, and this induced additional ADR
entrapment in a physical way. As a result, drug entrapment efficiency
was improved from 10 to 65%. In this process, drug entrapment was
further improved by desalting adriamycin in the presence of
triethylamine (TEA). ADR allows the micelle core more hydrophobic
so that more drug molecules can be entrapped in the core. ADR
molecules conjugated to the block copolymers stably disperse in a
high concentration with no precipitation, forming the polymeric
micelles. However, subsequent studies revealed that ADR formed
dimers in the presence of TEA through the head-to-tail reaction, and
these dimers indeed played an important role in micelle stabilization
[39]. Cytotoxicity studies have confirmed that ADR conjugated
covalently to the block copolymer has no bioactivity, indicating that
obtained PEG-p(Asp-ADR) block copolymers did not induce cytotoxi-
city but drug molecules physically entrapped in the micelle cores
suppressed cell viability. Therefore, it appeared that the drug release
rate would be a key factor to determine the cytotoxicity of the
polymeric micelles, and that drugs should be conjugated through
reversible binding linkers to regenerate active drugs for efficacy. It
must be noted that the micelle structure is stabilized when the
amount of ADR physically entrapped in the core increases. This
reduces systemic leakage of ADR while enhancing ADR accumulation
in the tumor tissue.
Solvents, ionic strength, temperature, and miscibility between
core-forming segments and drugs play an important role in the
process of micelle formation [40]. Amphiphilic block copolymers self-
assemble into polymeric micelles through a thermodynamic interac-
tion between the segments rearranging and forming domains. In these
domains, polymer segments with the same physicochemical proper-
ties are segregated into the most thermodynamically stabilized state
[41]. In addition to such self-assembling features, the easily-
772 Y. Bae, K. Kataoka / Advanced Drug Delivery Reviews 61 (2009) 768–784
Fig. 3. Micelle-forming polymer–drug conjugates synthesized in early studies.
modifiable chemical structure is another important advantage of the
polymeric micelles to give new functionalities to drug carriers.
Modification between the core and surface of the micelles is also
possible at the same time. Consequently the materials to be loaded can
be widely selected by optimizing molecular interactions in the core.
Nevertheless apparent physicochemical properties of the micelles
remain the same due to the hydrophilic PEG shell.
Driving force for micelle formation is mainly based on the
hydrophobic interaction, hydrogen bonding, metal complexation,
and electrostatic interaction [8]. In order to modify these properties,
functional groups are introduced to the core-forming segments.
Previous experiments have prompted reports that the polymeric
micelles from hydrophobic interactions remain stable in aqueous
solutions for days. Another method by which to prepare the polymeric
micelle involves the inducing of strong interactions between the core-
forming blocks, such as electrostatic interaction [42], and metal
complexation [12]. This method is based on the fact that the micelle
structure is formed by the nanosegregation of hetero-blocks of block
copolymers, and thus, micelles are spontaneously formed if a strong
interaction occurs between blocks forming the stable micelle core.
Basically, the reducing of free energy is the key factor in order to
prepare stable polymeric micelles. Therefore it must be emphasized
that entropy change is not always the major driving force for micelle
formation. When strong cohesive forces such as hydrogen bonding
and metal complexation are involved, enthalpy change plays a more
important role in stabilizing the polymeric micelles. Indeed the
polymeric micelles prepared solely through the entropy-driven
mechanism dissociate easily under the diluted conditions, and
therefore, these micelles are not suitable for parenteral drug delivery.
Size of polymeric micelles generally ranges from 20 to 100 nm,
which is clinically relevant for systemic drug delivery by the EPR effect.
Physicochemical evaluations showed these particles disperse stably in
the aqueous solutions with a good clinical relevancy. Core–shell
structure and size of micelles are similar to those of natural viruses. As
viruses protect the nucleic acid core with a protein-coated lipid
envelope, polymeric micelles sequester therapeutic agents from outer
environment in a hydrophobic inner core that is surrounded by a
hydrophilic outer shell. Optimized polymeric micelles stably disperse
hydrophobic drugs in aqueous solutions with no precipitation. Particle
size may change depending on the chemical structure of drugs and
type of micelle core. Solubility parameters between drugs and the
core-forming segment of block copolymers determine drug entrap-
ment efficiency. As described above, drugs can be covalently con-
jugated to the block copolymers to improve affinity between the drug
molecules and the polymers. The micelles with such chemical
modification show better stability in aqueous solutions. Stable micelles
can be sterilized by simply filtering the solutions. Obtained polymeric
micelles can be freeze-dried for a longer storage and reconstituted in
aqueous solutions prior to use. This provides a significant advantage
for pharmaceutical applications of the polymeric micelles.
Another significant property is that the polymeric micelles provide
different environments in the core, such as gel-like, amorphous and
crystalline environment. Such an environmental change in the core can
be utilized as a useful way to investigate the physicochemical properties
of the micelles. For example, when fluorescent probes like pyrenes were
incorporated in the micelles, fluorescence emission of pyrene excimers
and pyrene monomers change at 480 and 417 nm, respectively. It
provides information on the structure and dynamics of polymeric
micelles [35]. By monitoring the excimer-to-monomer ratio (Ie/Im or
I480/I417), one can determine the stability of the micelles. A similar
property is also observed when auto-fluorescent drugs are entrapped in
the micelles. High drug concentrations increased locally induce the
fluorescence quenching of drug molecules in the micelle core. Whereas
UV–VIS absorption is retained, physicochemical properties such as CMC
and distribution of the micelles accompanying drug release can be
observed by monitoring a change in fluorescence. The polymeric
micelles entrapping ADR exemplify these properties. Fluorescence of
ADR significantly decreases accompanying micelle formation while UV
absorbance maintains its value. This phenomenon is very useful in that
one can observe the distribution of micelles as well as their drug release
profile. Drugs released from the micelles recover their fluorescence, and
therefore, stability of the micelles and the fate of released drugs can be
directly observed by fluorescence microscopy [43].
3. pH-sensitive polymeric micelles with covalent drug conjugation
3.1. Drug conjugation via pH-labile hydrazone linkage
A new approach of drug loading to the micelles has been made
recently by conjugating drugs to the micelle-forming block copolymers
 Y. Bae, K. Kataoka / Advanced Drug Delivery Reviews 61 (2009) 768–784
through in vivo stimuli-responsive linkers [44]. In vivo stimuli may
include enzymes, oxygen and proton. Through micelle formation the
drug-binding linkers can be protected from the in vivo environment.
Since the micelle core has well-packed and limited space, enzyma-
tically degradable drug-binding linkers are less appropriate for the
design of environment-sensitive polymeric micelles. Previously, the
polymeric micelles appeared to effectively protect biological drugs
from enzymatical degradation [45]. Whereas protons, other ions and
small molecules in vivo that can penetrate into the micelle core easily
are considered effective triggers to initiate drug release from the
micelles. An approach of pH-controlled drug release is also useful to
target other in vivo acidic environments. Compared to physiological
conditions at pH 7.4, intratumoral space and intracellular compart-
ments, such as late endosomes and lysosomes, are known acidic
between pH 6.8–7.2 and pH 4–6, respectively. Thus, if drug carriers
could incorporate drugs stably at pH 7.4, and release the drugs at
pH below 6, these carriers would selectively deliver drugs to the
intracellular regions.
Schiff base, or azomethine, is considered the most facile and
appropriate linkage to design such pH-sensitive drug release systems.
Generally imine bond is stable at pH 7.4 while cleavable at pH below 6
[46]. However, the imine bond is reversible even at pH 7.4, it is
concerned that the conjugation between drugs and polymers are not
stable enough to retain drugs during blood circulation. To the contrary,
hydrazone bond appears to be more stable than the imine bond while
it shows excellent pH-sensitivity. More importantly hydrolysis rate of
hydrazone bond can be modulated further by introducing different
chemical groups adjacent to the hydrazone linkage [47]. In addition to
hydrolysis rate of hydrazone linkage itself, depending on the
environment where these hydrazone linkages are, drug release can
be controlled more precisely. For instance, if hydrazone bond is
sequestered from the outer environment, its hydrolysis would slow
down. On this basis the polymeric micelles with ADR conjugated to
the core-forming block through hydrazone bond have been developed
[43,44].
Fig. 4 shows the chemical structure of poly(ethylene glycol)-poly
(aspartate hydrazide) [PEG-p(Asp-Hyd)] block copolymers that
form pH-sensitive polymeric micelles. There are two synthetic routes
for this chemical modification. First approach is that PEG-PBLA is
deprotected to obtain PEG-p(Asp) block copolymers. Using isobutyl-
chloroformate, carbazic acid tert-butyl ester is introduced at the side
chain of PEG-p(Asp) in the presence of N-methyl morpholine. BOC
Fig. 4. Design of block copolymers for pH-sensitive polymeric micelles.
773
protecting groups are deprotected using TFA, and then hydrazide
groups are activated by removing salts [44]. Another approach is that
PEG-PBLA is reacted with hydrazine to give PEG-p(Asp-Hyd) through
a one-step ester-amide exchange reaction [48]. Compared to first
synthesis route, second synthesis route is more convenient in terms of
hydrazide group substitution yields. In addition the ester-amide
reaction allows one to control substitution rate from zero to 100% in a
quantitative way. It must be noticed that the substitution ratio be-
tween hydrazide and carboxyl groups plays a crucial role to maintain
the environment in the core to be proton-accessible but still hydro-
phobic. If the micelle core becomes hydrophilic, it is favorable for pH-
sensitivity but the micelles become unstable. On the other hand, if the
micelle core is too hydrophobic, drug molecules show extremely slow
release from the micelles. Studies showed 75–85% substitution of
hydrazide induced the optimal drug release pattern with clinically
relevant stability. Block copolymers with 100% substitution ratio often
perturb drug release due to too high hydrophobicity. They also may
form nano filaments other than micelles, increasing heterogeneous
carrier populations.
As shown in the figure, ADR is bound to the core-forming segments
of obtained PEG-p(Asp-Hyd) block copolymers through a hydrazone
bond. This enables the micelles to release drugs responding to the
intracellular pH change in the lysosomes (pH 4–5). Similar to other
drug-polymer conjugates, free drugs absorbed onto the polymers
through hydrophobic interaction should be removed completely.
Otherwise, the polymeric micelles will release drugs not only pH-
sensitively but also time-dependently in a mixed formulation. This
may require complicated analysis on drug action and distribution in
the determination of regimens. Mixed formulation of chemically-
conjugated and physically-entrapped drugs might provide better
activity. Yet on the other hand it could show drug leakage or toxicity
as seen in classical micelles. Drug release control is of importance
in order to adjust the amount of drugs that are actually given to the
cells after the delivery of therapeutic agents to the targeted site in the
body.
Drug release rate of pH-sensitive micelles is basically determined
by the hydrolysis rate of drug-binding linkers. Fig. 5 demonstrates that
drug release profile of the pH-sensitive micelles can be controlled to
take place selectively in the acidic conditions with pH below 6. These
conditions are optimized for the intracellular acidic environments
such as endosomes (pH 5–6) and lysosomes (pH 4–5). Drug release
studies are generally conducted by a dialysis method. Depending on
774 Y. Bae, K. Kataoka / Advanced Drug Delivery Reviews 61 (2009) 768–784

Fig. 5. Drug release patterns of pH-sensitive micelles under the closed and sink conditions.

how release experiments are conducted, there are two conditions for
drug release, which are ‘closed’ and ‘sink’ conditions. In the closed
condition, micelles undergo dialysis in the limited portion of outer
solutions. When mass transport reaches the equilibrium, drug release
may stop. Although it provides an estimated drug release profile, it
would not be a ‘real’ drug release profiles seen in the living cells or
other in vivo environments. For the drug release experiments in the
sink condition, micelles are put in a dialysis bag while outer solutions
keep being changed. In this way, inside and outside of the dialysis
bag do not reach the equilibrium, regenerating the conditions more
similar to the real physiological environments. Thus, experimental
setup is significantly important when it comes to release studies for
the polymeric micelles. Fig. 5 shows a difference in drug release
patterns of the micelles between the closed and sink conditions. It
clearly shows drug release is accelerated when the polymeric micelles
are incubated under the sink conditions. Although pH-dependent
drug release is shown in both conditions, it is obvious that drug
release reaches equilibrium in closed conditions. Whereas the poly-
meric micelles incubated in sink conditions keep releasing drugs.
These results implicate that drug release experiments should be
designed carefully considering the site of action for drug carriers.
In the meantime, time-dependent drug release patterns corre-
spond well with intracellular drug release of the polymeric micelles.
Cells take up large materials, like the polymeric micelles, by folding
the cell membrane inward and surrounding the materials to be
ingested in the forms of very small bubble-like endocytic vesicles. This
is called the endocytosis process that allows drug carriers to sneak
into intracellular regions avoiding cell membrane transporters. After
the polymeric micelles enter the cell through endocytosis, subsequent
mass transport takes place. The endocytic vesicles move from early
and late endosomes and finally to lysosomes where the proton con-
centration is 100 times higher than the physiological condition [49].
Consequently, it is of significance whether the micelle can precisely
function within living cells responding to the intracellular pH
decrease. Fig. 6 shows the images of cells exposed to the polymeric
micelles as well as free drugs. As seen in physical drug entrapment
type micelles, fluorescent drugs undergo fluorescence quenching in
the polymeric micelles core while showing strong fluorescence again
when drugs are liberated. Compared to the cells where free drugs
accumulate in the nuclei quickly after 1 h incubation, the cells treated
with the polymeric micelles still show the low fluorescence level after
the same incubation time. However, the cells with the polymeric
micelles show drug distributions in both cytoplasm and cell nuclei
after 24 h, while free drugs mainly accumulate in cell nuclei
irrespective of incubation time. It is intriguing that relatively large
amount of drugs remain in the cytoplasm when the cells are treated
with the polymeric micelles. This demonstrates the controlled and
sustained release of drugs from the polymeric micelles.

Fig. 6. Fluorescence quenching effects in the micelle core and intracellular distribution.
 Y. Bae, K. Kataoka / Advanced Drug Delivery Reviews 61 (2009) 768–784
3.2. Tissue penetration
Two decades ago, H. Maeda and Y. Matsumura proposed that
tumor-specific drug targeting can be achieved by macromolecules
through the EPR effect [50]. Despite extensive studies on DDS, tumor-
specific accumulation of macromolecules is still challenging because
there are several other substantial problems for macromolecular drug
carriers to overcome. Those problems are mainly related with low
concentrations of active drugs at the tumor tissues. It is due to the
limited accessibility of macromolecules into avascular tumor micro-
environments [51]. For these reasons, a particular attention has been
recently paid to the development of drug carriers that can deliver a
sufficient amount of active drugs to the deep into the tumor tissues
[52]. Previous preclinical and clinical studies have shown that func-
tional and structural features of drug carriers should be optimized
simultaneously in order to overcome the tissue penetration issue [53].
Prior to this study, it is important to confirm whether the polymeric
micelle can access the inside of the tumor tissue maintaining their
structures, or the polymeric micelles release drugs in the extracellular
region and released drugs penetrate into the tumor tissues. This issue
is crucial to cancer therapy using macromolecular drug carriers for the
treatment of solid tumors with limited vasculatures because it may
induce bystander effects of drugs.
In order to study the correlation between drug carrier design and
tissue penetration, reliable in vitro models are necessary. R. Suther-
land and coworkers adapted the methodology of cell aggregates to
cancer research for developing a suitable in vitro model of solid tumor
in 1971 [54]. Their method was to culture cells in a three-dimensional
structure, called multicellular tumor spheroids (MCTS), which was
pioneered by J. Holtfreter in the 1940s. Since then, interest in MCTS
has rapidly increased and expanded to other research areas like
biomedical science and basic cell biology, elucidating the roles of
microenvironmental factors in vivo [55]. Particularly, MCTS has
contributed to the understanding of cell response to various
chemotherapeutic treatment modalities, which provided an excellent
in vitro screening system for the studies of drug penetration, binding,
and action. It is known that cells in tumor microenvironment are less
sensitive to anticancer drugs. It is probably due to poor penetration of
drugs or other epigenetic variables. Therefore the drug concentration
should be increased by a factor that is highly variable according to the
Fig. 7. Multicellular tumor spheroid culture and in vitro tissue penetration study.
775
drug tested to obtain the same level of inhibition of cell growth
observed in vitro.
One of the most important characteristics of MCTS is a hetero-
geneous three-dimensional distribution of cells (Fig. 7). Although it
depends on the size, the cells located at the periphery of MCTS are
proliferating, and the cells in internal layers of MCTS are quiescent,
non-cycling, showing drug resistance. In the center of MCTS is a
necrotic core due to the deficiency of nutrients and oxygen. Several
techniques have been developed to prepare MCTS. The idea is to
culture cells by inhibiting cell adhesion on the cell culture plate. In a
traditional way, cell adhesion is inhibited by constant agitation.
Special culture plates become recently available, which are treated
with agar/agarose or extracellular matrix. Using these culture plates,
one can prepare MCTS by simply seeding cells under normal cell
culture conditions. Once the seeded cells form MCTS, they generally
continue to grow beyond confluence. MCTS are then sorted according
to the size for different experiments. The last possible technique for
MCTS culture is to use enzymes. By treating monolayered cells with
serum plasmin, cells can detach from the plate and grow as floating
MCTS. Two plasmin activators, urokinase plasminogen activator and
streptokinase, are known to induce the same results. Such growth
pattern is also shown when aged serum that is deficient in
plasminogen activator inhibitors was used for cell culture. However,
if fresh serum is added, these MCTS revert to monolayered cells. Some
hormone-dependent human cancer cells require specific constituents
for MCTS cultures.
Based on these rationales, tumor tissue permeability of the poly-
meric micelles was investigated using MCTS. In a previous study,
physical drug entrapment type micelles were incubated with MCTS
while free drugs and liposomes were used as control [56]. After certain
incubation time, thin cross-sectioned samples of MCTS were obtained
using cryomicrotome. Fluorescence microscopic observations showed
that drug molecules distributed throughout the MCTS when treated
with free drugs and polymeric micelles. MCTS treated with liposomes
showed limited area where drugs are accumulated. Although these
initial observations demonstrated the polymeric micelles show better
drug delivery efficiency compared to liposomes in terms of tissue
penetration, diffusion of small molecule drugs which are released from
the polymeric micelles cannot be ignored. It is because physical drug
entrapment type micelles release drug time-dependently, and thus,
776 Y. Bae, K. Kataoka / Advanced Drug Delivery Reviews 61 (2009) 768–784
aforementioned results do not implicate that the polymeric micelles
penetrate the MCTS with the intact structure. In order to answer this
question, MCTS was treated with the pH-sensitive micelles in a
subsequent study [43]. Optical sliced fluorescence images of MCTS
were taken with the confocal laser scanning microscopy (CLSM). The
images showed that drug distribution at the center of MCTS gradually
increased and all cells in the MCTS were exposed to drugs eventually.
The drug distribution pattern of pH-sensitive micelles was similar to
that of free drugs. However, only the MCTS treated with the pH-
sensitive micelles showed co-localization of drugs in the cytoplasm
and nuclei. This obviously demonstrates that polymers distributed
around the center of MCTS. Colocalization of drugs in the cytoplasm
and cell nuclei indicates that the pH-sensitive micelles can reach the
center of 200 μm MCTS and release drugs in the cells, realizing spatial
and temporal control of drug distribution. To the contrary, liposomes
showed no efficient tissue penetration. These surprising results should
be studied further because it is still unclear whether the polymeric
micelles can penetrate the tissues maintaining their nano core–shell
structures. Dissociation of polymer micelles might have been followed
by diffusion. Nevertheless, a possible explanation for such a dramatic
difference between the polymeric micelles and liposomes would be
made as the fact that the polymeric micelles have a flexible PEG shell
that can shrink into smaller size than their apparent hydrodynamic
diameters. This is a crucial difference between the polymeric micelles
and liposomes. Particle size of liposomes may remain stable because
they have the more robust vesicle structure.
These results are also intriguing in that the polymeric micelles can
penetrate the tissues easily than expected in spite of their high
molecular weight more than 2,000,000 as well as the hydrodynamic
radius larger than 25 nm. Literatures show polymers with up to
molecular weight of 70,000 effectively extravasate from vascular
compartment to tumor tissues [57]. Tissue penetration of macro-
molecules decreases significantly when molecular weight is higher
than 10,000. It is possibly due to the interaction of polymers with the
extracellular matrix. Notably, despite the slow rate, polymers with
molecular weight of 70,000 can penetrate into the tissues, demon-
strating a clear difference from macromolecules with molecular
weight of 2,000,000. Therefore hydrodynamic radius of macromole-
cules would play an important role in tissue penetration rather than
absolute molecular weight.
Polymers generally show limited tissue penetration. Molecular
weight is an important factor to determine the degree of penetration.
Yet studies show hydrodynamic radius might play a crucial role in
tissue penetration. Aforementioned studies using MCTS on tissue
Fig. 8. Synthesis of folate-conjugated pH-sensitive block copolymers for active targeting.
penetration partially demonstrate inter- and intracellular behaviors of
the polymeric micelles within avascular tumor tissues in the body,
which have not been revealed yet by the conventional in vitro cell
assays based on monolayered cell culture. Therefore, although the
mechanism of tissue penetration still needs to be elucidated, studies
show that the polymeric micelles are likely able to deliver drugs across
the tissues in comparison to other drug carriers.
4. Folate-conjugated polymeric micelles
4.1. Folic acid and active cancer targeting
As for cancer targeting methods, one might simply consider that
active targeting is more advanced than passive targeting. Yet active
targeting cannot be realized until passive targeting is properly
achieved. Even if newly designed drug carriers have great potential,
their intrinsic physicochemical properties such as solubility, particle
size, and surface properties still play an important role in determining
the fate in vivo [64]. Therefore it is of significant importance to select
appropriate interactions and molecules for active targeting, without
hampering physicochemical properties of original drug carriers.
From these aspects, targeting molecules are generally exploited to
enhance the uptake of macromolecules. These targeting molecules
encompass antibodies, truncated oligopeptides, and small molecules.
Large targeting molecules tend to result in undesirable intermolecular
interaction or aggregation. To the contrary, small molecules and
oligopeptides have drawn a particular attention because they would
have the least influence on the physicochemical properties of
macromolecular drug carriers. The only possible disadvantage of
small ligands is the binding and recognition efficiency with respect to
the receptors. Therefore, drug delivery systems with small targeting
molecules have become popular yet challenging to design active drug
targeting [58,59].
Among small targeting molecules, folic acid is known as a
promising candidate because of its low molecular weight (MW =
441.40) and excellent binding/recognition affinity against the recep-
tors called folate-binding proteins (FBP). Cancer cells overexpress FBPs
on the cell membrane which can be targeted by delivery systems using
folate [60–63]. Hetero bifunctional PEG is used for the surface
modification of the polymeric micelles [64]. Fig. 8 shows the synthesis
of folate-conjugated PEG-PAA block copolymers that form multi-
functional polymeric micelles featuring both pH-controlled drug
release and folate-mediated cancer targeting [65]. It must be noticed
that folic acid has two carboxyl groups at its α and γ positions. Studies
 Y. Bae, K. Kataoka / Advanced Drug Delivery Reviews 61 (2009) 768–784
show that only γ-position activated folate retains its activity, in terms
of receptor recognition [63]. Another report, however, demonstrates
that both α- and γ-position modified folates can interact with FBP
irrespective of α and γ positions [66]. Although controversial, it is
obvious that homogeneous compositions of folate conjugates are
preferable to evaluate the bioactivity and cellular interaction between
drug carriers and the targeted cells precisely.
In order to confirm receptor recognition, surface plasmon reso-
nance (SPR) measurements are widely used. As shown in Fig. 9, folate-
conjugated micelles were allowed to flow onto the gold surface
where FBPs are installed. This analysis clearly showed the interaction
between folate-conjugated polymeric micelles and FBPs. The folate-
conjugated micelles interact with FBPs even in a low concentration.
Interestingly obtained signals demonstrated that there is no sig-
nificant difference in receptor recognition as long as the polymeric
micelles have folate content more than 10% on their surface. It is
believed that the folate installed on the micelle surface interacts with
the receptors multivalently and therefore the interaction is more
efficient than a univalent interaction between small molecules.
Therefore, it is concluded that the modification of the micelle
surface with small molecule targeting molecules induces a minimum
change in surface properties while retaining receptor recognition. It is
noticeable that the micelles with low folate content still can recognize
FBPs. One of the most difficult things to design polymeric micelles for
active targeting may be the maintaining of the surface property.
Higher folate content might increase receptor recognition of the
polymeric micelles, yet it may also increase a risk of phagocytosis by
macrophages during the blood circulation. Data show that, when
folate content becomes close to 100%, the polymeric micelles undergo
precipitation at high concentrations. It demonstrates possible inter-
micellar aggregation due to the altered surface properties. Therefore,
the balance between passive and active targeting is a key factor to
achieve the highest efficacy of cancer targeted delivery.
Fig. 9. Receptor recognition and cellular interaction of folate-conjugated micelles.
777
4.2. Cellular interaction and bioactivity response
SPR measurements demonstrated that folate-conjugated micelles
recognize FBPs efficiently. Excellent receptor recognition with low folate
content is a great benefit. The cellular interaction and bioactive response
of folate-conjugated polymeric micelles were confirmed previously.
Human pharyngeal cancer cell-line KB cells overexpress FBPs on the
surface, providing a nice in vitro model. Studies showed folate instal-
lation increased cellular uptake of macromolecules. Folate-conjugated
polymeric micelles demonstrated comparable cellular interaction,
which is confirmed by flowcytometry (FCM) measurements (Fig. 9).
FCM showed an instant increase in cellular uptake of the polymeric
micelles, followed by a time-dependent increase of intracellular drug
accumulation. Micelles without folate were used as control yet did not
show any instant cellular interaction. The results are corresponding well
with the SPR results, potentiating cancer cell targeting of the folate-
conjugated polymeric micelles. Noticeably complete suppression of
non-specific interaction of the polymeric micelles with cancer cells is
unlikely because the cells with no receptors can engulf the polymeric
micelles through macropinocytosis. The installation of targeting
molecules is currently at a stage to improve cellular uptake. Cancer
cell-specific uptake of nanoparticles still remains a challenging issue.
Nevertheless, through active targeting, the polymeric micelles can
recognize cancer cells more efficiently than normal tissue and cells.
In order to confirm general effects of folate installation on anti-
tumor activity of the polymeric micelles, various human cancer cell
lines have been screened using the folate-conjugated polymeric
micelles. As shown in Fig. 10, most cancer cell lines become more
sensitive to folate-conjugated polymeric micelles than the polymeric
micelles without folate. In some cancer cells, bioactivity of folate-
conjugated polymeric micelles was even greater than free drugs.
Particularly the drug resistant HCT-15 cell line showed marked
sensitivity to the chemotherapy. Considering multidrug resistance
778 Y. Bae, K. Kataoka / Advanced Drug Delivery Reviews 61 (2009) 768–784
Fig. 10. Cytotoxicity response screening in various human cancers.
(MDR) is mainly induced by overexpression of P-glycoproteins on the
cancer cell surface, these results potentiate the treatment of other
MDR phenotype cancer cells using the polymeric micelles for active
drug targeting.
5. Antitumor activity and bioavailability
5.1. In vitro cytotoxicity
Most anticancer drugs are limited in their clinical applications
because of high toxicity and low water solubility. Toxicity is mainly
from the non-specific distribution of anticancer drugs in the body.
Serious problems stem from the fact that the drug concentration range
showing pharmaceutical activity without toxicity is extremely narrow,
also called the therapeutic window. Non-specific drug distribution
results in rapid clearance of therapeutic agents from the body. Such
rapid drug clearance results in repetitive drug infusion in order to
maintain drug concentrations in the blood. Repetitive drug adminis-
tration may induce either chronic toxicity or resistance to chemother-
apy. Pioneers in 1970s suggested that drug delivery systems based on
polymer science and supramolecular chemistry could overcome these
limitations [67]. Aforementioned approaches also have demonstrated
that drug efficacy is indeed enhanced by using drug carriers including
the polymeric micelles.
Cytotoxic activity of the polymeric micelles is subject to the drug
release patterns [68]. Physical drug entrapment type micelles release
drugs time-dependently right after incubation or even preparation.
The drugs released in the extracellular tumor regions can easily diffuse
into the cell. As a result, physical drug entrapment type micelles show
cytotoxicity as high as small molecule drugs. On the other hand, pH-
sensitive micelles release drugs selectively under acidic conditions
after intracellular uptake. Intracellular drug concentrations induced
by the pH-sensitive micelles, therefore, may be lower than that of
physical drug entrapment type micelles. This results in a clear
difference in cytotoxicity. It is of interest that cytotoxicity of the pH-
sensitive micelles increases time-dependently, demonstrating a
gradual increase in intracellular drug concentrations. Whereas, the
physical drug entrapment type micelles show effective cytotoxicity
even with short incubation time.
Cytotoxicity is also dependent on the incubation time. Therefore
when cells are incubated with the polymeric micelles for a longer
time, cytotoxicity of the polymeric micelles increases irrespective of
the drug-loading methods. Theoretically cytotoxicity of these different
micelle formulations would reach to the similar level of that of free
drugs. This means one can control the cytotoxicity level against the
targeted cells simply by selecting drug entrapment methods. Thus, the
correlation between drug release control and cytotoxicity is very im-
portant particularly when designing drug carriers for either systemic
or local drug delivery. For these reasons, in contrast to free drugs, in
vitro cytotoxicity of the polymeric micelles cannot be interpreted
directly to estimate the in vivo antitumor activity.
5.2. Biodistribution
In order to develop the polymeric micelles optimized for in vivo use,
the fate of polymer components and their assemblies in the body should
be studied. This enables us to design the polymeric micelles that safely
protect and deliver therapeutic materials in vivo avoiding the host
defense system. Generally, distribution of external materials in the body
is influenced by the surface properties. In comparison to low molecular
weight materials, polymers which hardly penetrate blood vessel walls
circulate in the vascular space after infusion. If polymers are cationic
they aggregate with anionic proteins in the body or directly interact with
cells, inducing pulmonary embolism for instance. If polymers are
sufficiently water soluble, they start circulating in the blood stream and
reach the first gate to pass, the kidneys. The kidneys excrete external
materials with molecular weight and size of less than 50,000 and 6 nm,
respectively, into the urine [57]. In addition to the renal filtration,
because the renal capillaries are negatively charged, polymers with
positive charges that manage to avoid being trapped in the lung are
filtered completely in the kidney. Thus renal filtration rate is significantly
different between the molecules with the same molecular weight
but different charge [51,52]. Even if macromolecules overcome both
pulmonary and renal filtration successfully, protein adsorption initiates
the immune response including phagocytosis of macrophages. Particu-
larly the reticuloendothelial systems (RES) remove these unfavorable
materials with a high efficiency. The RES is composed of monocytes
and macrophages located in the reticular connective tissues, such as the
liver and spleen. It is responsible for engulfing and removing cellular
debris, old cells, and unfavorable external materials from the blood.
Since protein adsorption occurs also by hydrophobic interactions,
bioavailability of drug carriers relies on how effectively the polymeric
micelles can sequester hydrophobic drug payloads from the in vivo
environments.
In early studies, physical drug entrapment type micelles appeared
to circulate in the blood stably and accumulate in the tumor tissues
 Y. Bae, K. Kataoka / Advanced Drug Delivery Reviews 61 (2009) 768–784 779

Fig. 11. Biodistribution of polymeric micelles.

selectively [69–71]. Fig. 11 shows that the pH-sensitive micelles also In the meantime, the polymer–drug carriers including the poly-
circulate in the blood for a long period of time, which was revealed by meric micelles would have another fundamental problem, which is
murine tumor model with human pharyngeal cancer cell KB. related to the total amount of polymers used for the delivery of
Compared to free drugs that underwent the body clearance within drugs enough for the treatments. The low drug-loading content
3 h, about 15–20% of total injected polymeric micelles still remained in requires a large amount of polymers to deliver active drug molecules
the blood after 24 h. Folate-conjugated polymeric micelles show an sufficient to antitumor activity. As certified by the U.S. Food and Drug
intriguing correlation between ligand content and tissue accumula- Administration (FDA), most polymers used for biomedical science are
tion. It is surprising that folate conjugation shows almost no change in biocompatible and non-toxic. Nevertheless, the effects of a large
tumor tissue accumulation of the polymeric micelles. Despite SPR amount of polymers on the long-term side effects are not revealed
measurements that showed a marked decrease in receptor recogni- completely and the answer is still controversial. Even from the clinical
tion with substitution below 10%, there was no noticeable change in point of view, injecting a large amount of polymers to the body is not
the biodistribution results. In contrast, the polymeric micelles with patient-friendly. Delivery efficiency in terms of the amount of the
higher folate content appeared to accumulate in the liver and spleen polymers is particularly serious when repetitive drug injections are
more preferentially. Folate conjugation seems to change the surface necessary for the treatments. Therefore, it is preferable to reduce the
property of the polymeric micelles, resulting in the increase amount of polymers not only because of better delivery efficiency but
accumulation in the liver and spleen. These results postulated that also because of preventing potential long-term side effects in the
active drug targeting may mainly improve the cellular interaction patients.
rather than alter the characteristic biodistribution of the polymeric
micelles, although this might be specific to folate-conjugated systems. 5.3. In vivo antitumor activity
When it comes to biodistribution data analysis, it is important to
understand that accumulation of the polymeric micelles does not Reduced toxicity and effective activity of physical drug entrapment
always mean the intracellular drug accumulation, and also that it is micelles were observed previously in animal models against all
not the amount of drug carriers that influence toxicity but that of free cancers tested such as mouse colon carcinoma (C26), mouse sarcoma
drugs released within the tissues. Normally drug concentrations are (M5076), human lung cancer (Lu-24), human breast cancer (MX-1)
proportional to the accumulation of prodrugs, yet in case of drug and mouse leukemia (P388) [72–76]. The pH-sensitive and folate-
carriers, drug release patterns should be considered carefully. This fact conjugated micelles also showed an improved activity and reduced
results in some discrepancy between biodistribution and toxicity data toxicity. In Fig. 12, time-dependent changes in tumor volume and body
of the polymeric micelles. For instance, although accumulation in the
liver and spleen increased in proportion to prolonged blood circula-
tion, the polymeric micelles generally show less toxicity than free
drugs. This result emphasizes again the importance of drug release
control along with cancer targeting approaches. For these reasons,
biodistribution studies should be conducted by considering that
accumulation of the polymeric micelles is not always the same with
the actual amount of active small molecules. Traditional biodistribu-
tion studies simply show accumulation of materials in the tissues. In
most cases, these materials are small molecules with excellent cell/
tissue penetration, and therefore, obtained data can be directly
converted into estimated toxicity against each tissue. In contrast,
biodistribution of macromolecular drug carriers only demonstrates
the level of the accumulation, providing no data that can be
interpreted directly to tissue toxicity. It is because the amount of
active drugs may remain low and increase time-dependently since
some drugs could be still conjugated to the polymeric micelles even if
they are accumulated in the tissue. Fig. 12. Antitumor activity of polymeric micelles.
780 Y. Bae, K. Kataoka / Advanced Drug Delivery Reviews 61 (2009) 768–784
weight are summarized. The animals were treated with free drugs
and the polymeric micelles. Human pharyngeal cancer KB cells
overexpressing folate receptors were used for evaluating the effects
of folate-mediated active targeting on antitumor activity [62]. Free
drugs show effective tumor growth suppression, yet they are
accompanied with serious body weight decrease of the animals. In
comparison, the polymeric micelles showed less toxicity, retaining
antitumor activity. Previously, physical drug entrapment type micelles
are as potent as free drugs in terms of the dosage used for the
treatment [73,74]. The pH-sensitive micelles were also beneficial in
suppressing tumor growth, yet the dose was slightly higher than that
of physical drug entrapment type micelles as well as free drugs.
Aforementioned pharmacokinetic profiles possibly explain why
pH-controlled drug delivery systems using micelles require a higher
dose. It is unlikely there is a notable difference in tumor accumulation
efficiency between physical entrapment type and pH-sensitive
micelles. Thus, it is postulated that drug release profile and its followed
drug diffusion within the tumor tissue may induce such differences.
Physical entrapment type micelles in early studies can release drugs in
the blood as well as extratumoral compartments. After drug molecules
are released from the micelles, they freely diffuse into the tumor
tissue, showing the same path of mass transport for small molecule
drugs. To the contrary, pH-sensitive micelles need to be localized in the
intracellular acidic compartments such as endosomes and lysosomes
to initiate drug release as described above. Although there might be a
bystander effect, drug distribution of controlled delivery systems is
mainly determined by tissue permeability of carriers, which is lower
than small molecule drugs. As a result, more pH-sensitive micelles are
necessary to achieve the same antitumor activity compared to free
drugs and physical drug entrapment type micelles.
The pH-sensitive micelle is characterized by the lowest toxicity
among the formulations, taking advantage of controlled drug release
systems that can reduce non-specific drug distribution in the body.
Indeed, biodistribution studies consistently showed that micelles
accumulated in the liver and spleen [68–71]. Considering low toxicity
of the micelles, biodistribution results do not correspond well with
antitumor activity. Although antitumor activity of pH-sensitive
micelles is slightly lower than physical drug entrapment micelles, a
difference in toxicity between different drug-loading micelle for-
mulations demonstrates that the controlled release systems would be
more promising in terms of lowering side effects while retaining
activity. In addition, active targeting increases in vivo activity of the
micelles, although biodistribution studies do not show a significant
difference with and without folate. The most probable hypothesis
on this self-discrepancy is that the micelles are removed from these
tissues before they enter the cells and start releasing drugs inducing
toxicity. Macromolecules that avoid renal clearance are known to be
excreted from the body through the bile duct. Polymeric micelles
might follow the same fate in the body. This is the reason why in vivo
activity and biodistribution of environment-sensitive micelles do not
always correspond with each other. Recent pathological observations
on the liver tissue from the animal treated with pH-sensitive micelles
show that intracellular accumulation of the micelles in hepatocytes is
extremely low [77]. On the other hand, small molecule drugs accu-
mulate largely in hepatocytes. Therefore, differential cellular interac-
tion and metabolism of the micelles are also considered the reasons
for low toxicity. Further investigations should elucidate the metabolic
mechanism of the micelles.
The balance between antitumor activity and toxicity determines
effective regimens and drug formulations. The therapeutic window
gives a clue how one can keep balancing efficacy and toxicity.
Accumulating data show that the polymeric micelles are superior to
conventional small molecule drug formulations. Fig. 13 summarizes
the maximum tolerated dose (MTD), effective dose (ED) and
treatment-to-control (T/C) ratio of free drugs, physical drug entrap-
ment type micelles, pH-sensitive micelles and folate-conjugated
Fig. 13. Comparison of toxic dose (TD) and effective dose (ED50) between drug
formulations.
micelles. ADR was effective at 10 mg/kg, yet the animals underwent
serious toxicity at 15 mg/kg. In contrast to free drugs, the pH-sensitive
micelles effectively suppressed tumor growth in xenografted mice
with relieved toxicity. The pH-sensitive polymeric micelles were
safely injectable up to 40 mg/kg dose, and the tumors were com-
pletely disappeared in three of six mice at 20 mg/kg. These results
indicate that the bioavailability of the loaded drugs is remarkably
improved by intracellular pH-sensitive drug release control.
Meanwhile, MTD increased about 4-fold or more while ED was
between 20 mg/kg and 40 mg/kg. In case of folate-conjugated
micelles, tumor-bearing mice were cured at 7.5 mg/kg dose, which is
even lower than ED of free drugs. These results are intriguing in that
active drug targeting does not change biodistribution of the polymeric
micelles but remarkably improve the bioactivity. Comparing the width
of dose between TD and ED50 (a dose for the complete treatment of
50% animals), all micelle formulations show the broader therapeutic
windows than the free drug formulation. It indicates that the
polymeric micelles can be safely injected for the treatment over the
broad dose range with effective antitumor activity and low toxicity,
possibly reducing the number of injection.
6. Combination chemotherapy using polymeric micelles
6.1. Modification of delivery environment
It is obvious that the polymeric micelles significantly improve the
efficacy and reduce toxicity, compared to the conventional drug
formulations. Animal studies also demonstrate that antitumor activity
of the polymeric micelles is directly influenced by the altered PK
profiles. Interestingly, in case of folate-conjugated polymeric micelles,
antitumor activity increased significantly although PK profiles did not
markedly change compared to the polymeric micelles without folate.
This demonstrates that the success of cancer chemotherapy may be
determined mainly by how the polymeric micelles interact with
cancer cells after accumulation. Therefore, the study on drug delivery
environments would be as important as the design of drug carriers to
achieve successful cancer therapy.
Indeed chemotherapy using drug carriers often suffers from tumor
tissue permeability due to the limited vasculature and thick fibrosis.
Pancreatic and schirrous carcinoma are such examples, which have
not been treatable through conventional small molecule drug formu-
lations. As one of the possible approaches to treat these refractory
cancers, combination chemotherapy using the pH-sensitive micelles
and intracellular signal inhibitors has been recently investigated [77].
The concept is to modify delivery environment to be more suitable to
 Y. Bae, K. Kataoka / Advanced Drug Delivery Reviews 61 (2009) 768–784
Fig. 14. Combination of targeted micelle delivery with TGF-β signal inhibition.
drug carriers and thereby therapeutic efficacy can be improved. In this
regard, blood vessels are probably the most useful target, regulating
influx and efflux of drug carriers as well as payloads to the tumor
tissues.
There are two approaches to utilize the blood vessels. One is to take
advantage of abnormal tumor vasculature and the other is to
normalize tumor blood vessels. Among these, utilizing abnormality
of tumor vasculatures has drawn an attention because the growing tip
of tumor blood vessel can be temporarily opened by inhibiting a
transforming growth factor β (TGF-β) signal pathway (Fig. 14). After
around 24 h, however, these blood vessels are closed again. This helps
entrap the polymeric micelles in the tumor tissues. By modifying the
delivery environment, antitumor activity of the polymeric micelles
appeared to significantly increase. Compared to control, the combina-
tion treatment of the pH-sensitive micelles and TGF-β inhibitors
remarkably suppress tumor growth of pancreatic as well as schirrous
gastric cancers. TGF-β inhibitors are initially developed as anticancer
drugs to suppress tumor growth. The TGF-β signal pathway is
characterized by its Janus-like functions that inhibit tumor growth
at the early disease stage but enhance cancer metastasis at the late
disease stage. Significance of the aforementioned combination
method is that this combination therapy requires only a small amount
of TGF-β inhibitors below the concentrations showing side effects.
This new class of approach would be promising in cancer combination
therapy using macromolecular drug carriers.
Certainly it would be also an effective approach to normalize
tumor vasculatures in order to improve the distribution of macro-
molecular drug carriers. However, it is still controversial whether the
normalization of tumor blood vessels influences the EPR effects,
tumor metastasis, and drug efflux and metabolism. Although the
methods for tumor vasculature normalization is not covered in this
review, obtained results and previous literatures support that the
modification of tumor environment is crucially important to achieve
more efficient cancer treatment using drug carriers [78].
Fig. 15. Concurrent drug delivery using mixed micelles for intelligent drug combination therapy.
781
6.2. Concurrent delivery of multiple therapeutic agents
From the pharmaceutical point of view, concurrent infusion of
multiple drugs is effective for the treatments yet extremely difficult to
realize because every drug shows distinct pharmacokinetics and
comes with various risks accompanying co-action of another drug
molecule. Solely determining a formulation for co-solubilization of
multiple drugs requires enormous efforts because each drug has
different solubility parameters and miscibility [79]. DMSO, Cremophor
and other surfactants are generally used to dissolve poorly water-
soluble drugs. However, drug formulations using these vehicles have
inherent toxicity, and therefore only a limited amount is allowed for
infusion. More seriously drug molecules can interact with each other
inducing unfavorable side effects in this formulation. In conventional
formulation studies, extensive regimen scheduling might be able to
resolve the issue. Yet the patients should be repeatedly injected with
drugs at a certain interval to prevent possible toxicity due to the
interaction between drug molecules. These facts postulate that con-
current drug infusion and delivery might be achieved only by DDS
technology.
Recent efforts have revealed that the polymeric micelles would be
a promising drug formulation for the simultaneous delivery of
multiple drugs. By incorporating multiple drug molecules in the
same micelle core, one can concurrently deliver various therapeutic
agents to the tumor tissue with the identical PK profile. This approach
would also allow each drug incorporated in the same micelle to
function against distinct intracellular targets such as receptors,
proteins, organelles, mitochondria and DNA at the same time. Through
such flexible combination formulations, significant synergistic effects
are expected on cancer therapy [80]. These systems are being studied
called mixed micelles. The mixed micelles can be grouped into two
categories, which are ‘chemically mixed micelles’ and ‘physically
mixed micelles’ (Fig. 15). Chemically mixed micelles mean that
multiple drugs are conjugated to the same polymer chains and these
782 Y. Bae, K. Kataoka / Advanced Drug Delivery Reviews 61 (2009) 768–784
polymers form a single type of micelle. In comparison, physically
mixed micelles mean that one drug is conjugated to polymer A and
another drug is conjugated to polymer B, and these polymers A and B
form micelles A and B separately. Both micelle A and micelle B are then
mixed in a certain mixing ratio to prepare physically mixed micelles. It
must be noticed that physically mixed micelles are different from a
physical mixture of distinct micelles in that mixing ratio of multiple
drugs in the core can be fixed in the micelle solution. Considering
dynamic interactions between the polymeric micelles as in previous
chapters, if different micelles are physically mixed in the same
solution and incubated for a certain period of time, these micelles will
eventually form physically mixed micelles owing to chain exchange.
However, compared to the LMWS micelles, the polymeric micelles are
expected to suppress the chain exchange. Such time-dependent
changes in the micelle mixing ratios can be controlled by drug
conjugation and chemical modification of the micelle core. Prelimin-
ary data have shown that the mixing of physical drug entrapment type
micelles is much faster than that of covalent drug conjugation type
micelles.
One example for chemically mixed micelles has recently appeared,
showing that moderate synergistic combination effects [81]. DOX and
Wortmannin (WOR) are simultaneously conjugated to the micelle-
forming block copolymer for the concurrent inhibition of topoisome-
rase II and phosphoinositide 3, respectively. The mixing ratio between
DOX and WOR can be precisely controlled while the micelle size and
its distribution remain unchanged. Interestingly, physically mixed
micelles show a broader distribution with a larger particle size. It is
speculated that DOX and WOR are not miscible well in the same core,
and therefore the polymeric micelles have a loosen core. In both cases,
however, the polymeric micelles show similar cytotoxic activity with a
moderate synergistic effect. Moderate synergistic effect was probably
due to the partial degradation of unstable furan ring of WOR. Although
further studies are required, these new types of mixed micelle formu-
lations are intriguing in terms of the design of novel drug carriers
and would provide a promising approach for combination cancer
chemotherapy using various potent drugs concurrently.
7. Future prospects
Amphiphilic block copolymers comprising of hydrophilic and
hydrophobic segments are known to form self-assemblies such as
lamellas, vesicles, and micelles. The balance between hydrophilic and
hydrophobic segments determines the structures of these self-
assemblies. Among them, polymeric micelles have drawn significant
attentions in the field of drug delivery science in that: 1) the poly-
meric micelles have excellent stability in low concentrations; 2) the
polymeric micelles have clinically relevant particle size for systemic
drug delivery; 3) the hydrophobic core of the polymeric micelles
provides a nano depot for hydrophobic drugs sequestered from the
aqueous solution; 4) the surface of the polymeric micelles can be used
for the installation of targeting molecules achieving active cancer
targeting; and 5) multiple drugs can be incorporated in the same
micelles concurrently for effective combination therapy.
The polymeric micelles might be considered simply ‘carriers’ that
deliver therapeutic payloads from one place to another. However, as
described above, our previous studies on the classical and intelligent
polymeric micelles for the last two decades have clearly shown that
drug carriers as well as the polymeric micelles would be indeed a new
class of drugs that has unique PK/PD profiles in combination with
several bioactive materials. Therefore, the polymeric micelles along
with other drug carriers such as polymer–drug conjugates, dendri-
mers, and liposomes are expected to be a useful tool to realize the
most effective patient-friendly chemotherapy for the better quality of
life.
Nevertheless, a large number of questions still remain to be
answered. One of them might be the stability issue of the polymeric
micelles. Stability of the polymeric micelles is determined by the
methods and compositions that keep the self-assembling components
thermodynamically frozen above the CMC until these micelles deliver
and release bioactive payloads to the site of action. Such stability holds
a key of success to achieve effective cancer treatments. Scientists have
recently reported that the polymeric micelles are not always stable in
the body depending on polymer compositions and environmental
factors. For instance, polymeric micelles from PEG-polylactide seem to
release drugs mostly extracellular space rather than deliver drugs
stably to the intracellular space [82]. This is contradictory to the com-
mon understanding that polymeric micelles transport drug directly to
the cytoplasm through endocytosis. Therefore, it is too early to draw a
general conclusion from one particular example regarding in vivo
performance of the polymeric micelles. From this aspect, it would be
getting important more and more to conduct systemic study to clarify
the correlation between structural stability and bioactive function of
the polymeric micelles in the future, elucidating metabolism and
micelle transport across in vivo barriers [6].
Finally the dawning of combination therapy using polymeric
micelles is of significance. It might be an approach to install target-
specific molecules on the micelle, to modify drug delivery environ-
ments, or to incorporate different types of bioactives concurrently. In
every case, it is obvious that intelligent drug delivery using the poly-
meric micelles will possibly bring the most facile, versatile, and effi-
cient methodology in chemotherapy treatments for human diseases.
Acknowledgment
Authors thank Dr. Takao Yamori, Division of Molecular Pharmacol-
ogy, Cancer Chemotherapy Center, Japanese Foundation for Cancer
Research, for generating data shown in Fig. 10 through drug screening
method using human cancer cell-line panel.
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