PEGylated Polyester-Based Nanoncologicals

Abstract

Several PEGylated polyester-based nanoncologicals have been proposed in the literature, some of them nowadays being under preclinical/clinical trials or marketed. In this review, we describe the main features of PEGylated polyesters and their correspondent nanocarriers. A first part is devoted to intravenously injectable PEGylated nanocarriers, which represent the systems most investigated so far. After describing fundamental design rules dictated by the administration route, PEGylated nanocarriers currently under preclinical/clinical investigation or in the market will be described from a technological point of view and related therapeutic implications discussed. Finally, new perspective of use of PEGylated nanocarriers for oral and pulmonary delivery of anticancer drugs will be considered.

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Current Topics in Medicinal Chemistry, 2014, 14, 000-000 1
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Pegylated Polyester-Based Nanoncologicals
Claudia Conte1, Ivana d’Angelo2, Agnese Miro1, Francesca Ungaro1 and Fabiana Quaglia1,*
1Laboratory of Drug Delivery, Department of Pharmacy, University of Napoli Federico II, Via Domenico Montesano
49, 80131, Napoli, Italy; 2Di.S.T.A.Bi.F., Second University of Napoli, Via Vivaldi 43, 81100 Caserta, Italy
Abstract: Several PEGylated polyester-based nanoncologicals have been proposed in the literature, some of them nowa-
days being under preclinical/clinical trials or marketed. In this review, we describe the main features of PEGylated polyes-
ters and their correspondent nanocarriers. A first part is devoted to intravenously injectable PEGylated nanocarriers,
which represent the systems most investigated so far. After describing fundamental design rules dictated by the admini-
stration route, PEGylated nanocarriers currently under preclinical/clinical investigation or in the market will be described
from a technological point of view and related therapeutic implications discussed. Finally, new perspective of use of PE-
Gylated nanocarriers for oral and pulmonary delivery of anticancer drugs will be considered.
Keywords: Cancer, chemotherapeutics, drug targeting, nanoparticles, nanotechnology, polyethylenglycol, polymers.
1. INTRODUCTION
Cancer is a lethal disorder characterized by the develop-
ment of abnormalities in cells causing uncontrollable and
fast growth/division due to a combination of mutations. Be-
sides, tumors attain further support through interactions with
surrounding stromal cells, promoting their angiogenesis,
tissue invasion and metastasis to distant organs, along with
evasion from immune detection, thus rendering cancer
treatment very challenging. Although many advances have
been achieved in conventional chemotherapy, bioaccessibil-
ity of anticancer drugs to tumor tissue remains limited, large
doses are required, leading to high toxicity to normal cells
along with an increased incidence of Multi Drug Resistance
(MDR) [1]. Therefore, a growing interest has been devoted
to the development of novel strategies for the optimization of
the biodistribution of anticancer drugs in the body in order to
improve their efficacy and selectivity.
Amid novel approaches, delivery of chemotherapeutics
through nanoscale carriers (i.e. nanoncologicals) is nowa-
days considered one of the most promising research area [2-
7]. It is well recognized that nanoncologicals may allow cir-
cumventing the major shortcomings of conventional chemo-
therapy. In fact, the main clinical implication of nanomedici-
nes for cancer is related to their potential to ameliorate drug
toxicity profile, due to targeting/killing cells of diseased tis-
sues/organs while affecting as few healthy cells as possible.
To this purpose, proper design of the system has to take into
account not only peculiar drug properties (solubility, stabil-
ity, target location) but also i) tumor type; ii) tumor stage; iii)
final administration route [8].
So far, intravenous nanoncologicals have been the most
extensively studied systems, likely due to the possibility to
get access to solid tumors with different location in the body
*Address correspondence to this author at the Department of Pharmacy, Via
D. Montesano 49, 80131 Napoli – Italy; Tel/Fax: +39 81 678707;
E-mail: quaglia@unina.it
as well as the need of replacing highly toxic vehicles em-
ployed to deliver poorly soluble anticancer drugs. The main
mechanism underlying accumulation of nanoncologicals in
solid tumors is considered the presence of disfunctional
tumor vasculature and poor lymphatic drainage in tumor
tissues [9]. Architectural defectiveness and high degree of
vascular density generate abnormal tumor vessels that are
“leaky”, owing to basement membrane abnormalities and a
decreased number of pericytes lining the rapidly proliferat-
ing endothelial cells. Moreover, solid tumors are character-
ized by an impaired and lack lymphatic network that de-
creases the clearance of nano-sized carriers giving, conse-
quently, prolonged retention in tumor interstitium. This
effect indicated as Enhanced permeability and retention
effect (EPR) allows long-circulating nanoncologicals - that
is nano-sized carriers able to escape clearance operated by
Mononuclear Phagocyte System (MPS) and renal filtration-
to accu mulate in solid tumors by extravasation through
defective blood vessels (passive targeting) and to remain in
contact with tumor cells [9, 10]. Finely tuning of carrier
composition, size, surface properties and drug release rate
can properly modulate the biodistribution of a nanon-
cological in the body and, in turn, optimize therapeutic re-
sponse [11, 12].
Decoration of the carrier surface by covalently linked
polyethylenglycol (PEG) chains, the so called “PEGylation”,
is recognized as an effective strategy to increase nanon-
cological half-life in blood circulation and promote accumu-
lation in solid tumors [13]. PEG conjugation may offer other
important beneficial outcomes, such as reducing intermo-
lecular interactions and promotion of colloidal stability, as
well as enabling further surface-engineering of the system.
Actually, decoration with specific ligands able to interact
with cell surface structures overexpressed in cancer cells or
endothelial cell of tumor vessels is a fascinating option to
promote active accumulation of nanoncologicals predomi-
nantly in cancer tissues [6, 14].

2 Current Topics in Medicinal Chemistry, 2014, Vo l. 14, No. 9 Conte et al.
Several natural and synthetic materials have been inves-
tigated as well as employed to build up engineered PEGy-
lated nanoncologicals for cancer treatment, including lipids,
polymers and inorganic materials [7, 15]. Amid PEGylated
polymers currently under investigation for cancer therapy,
those based on polyesters, such as poly(D,L-lactide)
(PDLLA), poly(L-lactide) (PLLA), poly(lactide-co-
glycolide) (PLGA) and poly(-caprolactone) (PCL), cover a
relevant area in the field due to several advantages. Indeed,
these polyesters have been FDA approved for injectable
formulations for many years now, and demonstrated a safe
toxicity profile. PEGylation of polyesters has been consid-
ered as a valuable strategy to obtain copolymers combining
the useful characteristics of PEG and biodegradable polyes-
ters. These copolymers consist of biodegradable hard polyes-
ter blocks and soft flexible PEG segments, showing am-
phiphilicity, biocompatibility and biodegradability. They can
form a large variety of PEGylated nanostructures able to
entrap hydrophilic and hydrophobic drugs.
A remarkable property of polyester-based systems re-
sides in their ability to sustain the release rate of the en-
trapped drug, which can be highly beneficial to reduce the
number of administrations and resemble a metronomic ther-
apy of cancer (sub-therapeutic doses for long time frames). It
has been suggested also that drug accumulation and slow
release inside tumor cells can be useful to circumvent MDR
[1, 16, 17].
In this rev iew, we describe the main features of PEGy-
lated polyesters and their formulation as nanostructured sys-
tems. A first part is devoted to intravenously injectable PE-
Gylated nanoncologicals, which represent the systems most
investigated so far. Then, new implications of the use of
PEGylated nanosized carriers for oral and pulmonary deliv-
ery of anticancer drugs will be considered. After describing
fundamental design rules dictated by the administration
route, PEGylated nanoncologicals in the market or currently
under investigation will be described from a technological
point of view and related therapeutic implications discussed.
2. FORMULATION OF PEGYLATED POLYESTER-
BASED NANOCARRIERS
2.1. PEGylated Polyesters
PEGylation is recognized nowadays as a general strategy
to prolong blood circulation of nanocarriers (NCs) through
decreased uptake by the MPS, diminished enzymatic degra-
dation, and reduced renal filtration [13, 18]. By this way,
carrier accumulation in solid tumors through the EPR effet
can be envisaged[9]. Nevertheless, this technology may also
contribute (a) to reduce particle aggregation in colloidal dis-
persion and, in so doing, to increase NC stability; (b) to
modulate the hydrophilicity of the polymer, thus tuning drug
release rate; (c) to modify the surface of nanosized carriers
with specific moieties, possibly increasing the specificity of
anticancer drugs toward their target; (d) to achieve self-
assembling systems able to accommodate hydrophilic and
lipophilic drugs in their interior. Chemical versatility is cer-
tainly a great advantage of PEGylated materials [18]. Indeed,
the possibility to tune PEG molecular weight as well as ar-
rangement of PEG segments on carrier surface (i.e., PEG
cloud configuration) has allowed the building of a wide
range of structures designed ad hoc for a particular therapeu-
tic application.
Due to their established potential as diagnostic and thera-
peutic tools, nanoconcologicals made of PEGylated polyes-
ters are especially emerging in the field of cancer nanotech-
nology. Indeed, polyesters, such as PDLLA, PLLA, PLGA
and PCL, remain the most interesting polymers for this ap-
plication due to their biocompatibility and safety profile
[19]. In fact, the extensive biodegradation of polyesters,
through chemical or enzymatic hydrolysis of the ester bonds
to water-soluble low molecular weight compounds that enter
the normal metabolic pathways of the organism, allows the
development of fully biocompatible nanoncologicals. A key
element to consider for drug delivery application is the rate
at which PEG polyesters degrade in the body, that generally
depends on polyester properties. As a general rule, molecular
weight (commonly referret to as weight average molecular
weight – Mw – or number average molecula weight – Mn)
and crystallinity dictate degradation rate, with more hydro-
philic and shorter chains degrading at a faster rate (Table 1).
The rather high crystallinity of the polyesters based on only
one type of monomer, such as PCL or PLLA, results in slow
biodegradability and sometimes offer low loading of drugs
(which accommodate in the amorphous regions) and poorly
modulated release rate [19-21].
PEG-polyesters are composed of regions that have oppo-
site affinities for an aqueous solvent and, at proper building
block chemistry, molecular weight, hydrophilic/hydrophobic
balance, can form spontaneously supramolecular aggregates
of different shape and structure in aqueous phases. On the
other hand, different preparation techniques can allow for-
mation of core-shell NCs where a cloud of PEG chains is
formed on the surface. The resultant PEGylated NCs ben efit
chemical flexibility of amphiphilic block copolymers, which
allows their engineering as a function of the physico-
chemical properties of the incorporated drug, patho-
physiology of the disease, location of drug action site and
proposed route of administration. Finally, these polyesters
can be further engineered at PEG side through the chemical
functionalization of several motifs, including targeting
ligands, antibodies, peptides, fluorescent probes, imaging
agents and magnetic materials [22-24]. Therefore, simple
polyesters can turn into multifunctional elements suitable for
the development of advanced nanoncologicals for anticancer
drug delivery.
Semithelechelic monomethoxy PEG (mPEG) spanning
from 400 Da to 10 KDa is generally used to prepare pas-
sively targeted nanoncologicals, whereas different chemical
approaches are employed to obtain PEGylated NCs exposing
at surface specific ligands [14]. Among PEG polyesters,
PEG–PLLA or PEG-PDLLA block copolymers are am-
phiphilic polymers that can be synthetized in different mo-
lecular weight ranges and PEG/polyester ratios by ring-
opening polymerization between PEG or its derivatives (e.g.
mPEG or functionalized PEG) and lactide [21]. Block co-
polymers consisting of PCL (A) and PEG (B) segments with
different architectures, spanning from simple AB (diblock),
ABA or BAB (triblock), multiblock (star-shaped and graft)
have been synthesized analogously or with more or less
complex synthetic strategies [20]. As compared to

Pegylated Polyester-Based Na noncologicals Current Topics in Medicinal Chemistry, 2014, Vol. 14, No. 9 3
Table 1. Key features of main synthetic polyesters and PEG/polyesters co-polymers for design of PEGylated nanocarriers. Data
from [19-21, 25, 165, 166].
Polymer Water solubility
Molecular
weight
Molecular ar-
rangement
Glass transi-
tion tempera-
ture (Tg)
Melting
point
Degradation
mechanism Degradation rate
PCL Highly hydrophobic 3,000-
90,000 Da
Semicrystalline
(varying with
polymer Mw)
-60°C 55-60°C
Random non-
enzymatic cleavage
followed by enzy-
matic fragmentation
Rather slow (up to 2-3
years)
PLA Very hydrophobic
(PGA<PLLA<PCL)
20,000-
120,000
Da
Semycristalline
(PLLA) or
amorphous
(PDLLA)
60-65°C 170-
190°C
Bulk erosion-
diffusion degrading
polymer (acidic
degradation prod-
ucts)
PLLA can take 2-5
years for complete
resorption
PLGA
Hydrophobic
(higher as LA
amount increases)
7,000-
250,000
Da
Amorphous (due
to random dis-
tribution of L-
and D-lactide
monomers)
55-60°C N/A
Bulk erosion-
diffusion degrading
polymer (heteroge-
neous erosion, faster
in the centre)
From weeks to months
(function of Mw and
LA:GA ratio, with the
fastest degradation for
PLGAs 50:50)
mPEG-PCL
Amphiphilic poly-
mers (hydrophilic-
ity depends on
PEG/PCL ratio)
2,000-
60,000 Da
Semicrystalline
(crystallinity
depends on
PCL/PEG ratio)
-60/-20°C 70-85°C
Passive hydrolysis
of ester bounds and
enzymatic fragmen-
tation
Faster than PCL
(higher as PEG amount
increases)
mPEG-PLA
Amphiphilic poly-
mers (hydrophilic-
ity depends on
PEG/PLA ratio)
2,000-
60,000 Da Amorphous
40-45°C (re-
duction of
PLA Tg)
N/A
Bulk erosion-
diffusion degrading
polymer
Faster degradation rate
than PLLA.
mPEG-PLGA
Amphiphilic poly-
mers (copolymers
with low Mw
and/or high
PEG/PLGA ratio
are water-soluble)
2,000-
60,000 Da Amorphous
35-40°C (re-
duction of
PLGA Tg)
N/A
Bulk erosion-
diffusion degrading
polymer
Faster than PLGA
(degradation/erosion
mechanism without
any lag time)
Abbreviations: PCL = poly(-caprolactone); PLA = poly(lactic acid); PLGA = poly( lactide-co-glycolide); mPEG = methossy-PEG.
PEG-PLA, copolymers comprising PEG and PLGA blocks
are more hydrophilic and considered more suitable than
their bare PLGA counterpart for the delivery of hydro-
philic macromolecules [25]. On the other hand, PEG-PCL
copolymers often present the same crystalline structure of
the corresponding bare PCL. However, due to its marked
lipophilic character, PCL is considered an excellent mate-
rial to prepare self-assembling systems, being prone to
form a stable solid-like lipophilic core where poorly wa-
ter-soluble drugs can be entrapped [20] .
2.2. General Features of PEGylated Nanocarriers
Amphiphilic PEG-polyesters can form a large variety
of core-shell NCs, such as nanoparticles (NPs) [26, 27],
micelles [28-30], vesicles called polymersomes [31-33],
depending on the properties of the base material and the
preparation method [14, 34] (Fig. 1). The drug to deliver
can be adsorbed on NC surface or, more often, encapsu-
lated inside the polymer core, as well as covalently linked
to the polymer (this last strategy will not be treated in this
review).
The general term “nanoparticles” refers to two sub-
categories of nanosystems with different organization named
nanocapsules and nanospheres. Nanocapsules are formed by
a polymer shell surrounding a liquid or a solid core at room
temperature [27]. The liquid core can be either oily, hence
allowing a high payload of hydrophobic drugs, or aqueous,
enabling encapsulation of water-soluble drugs. Nanospheres
are matrix particles where the entire mass is solid and con-
tains drug interspersed or dissolved in the polymer [4].
Taking advantage of the amphiphilic properties of the
polymer, perfect micelles with a polyester core and a PEG
shell can be formed in aqueous solution [34]. The formation
of micelles is driven by the decrease of free energy in the
system because of the removal of hydrophobic fragments
from the aqueous environment and the re-establishing of
hydrogen bond network in water [35]. Hydrophobic seg-
ments of amphiphilic molecules form the core of a micelle,
while hydrophilic fragments form the micelle shell. These
systems present the typical core-shell structure and preferen-
tially encapsulate lipophilic drugs in the polyester core
whereas shell acts as a stabilizer of the colloidal system and

4 Current Topics in Medicinal Chemistry, 2014, Vo l. 14, No. 9 Conte et al.
as biointerface with the body [15, 35]. Micelles are charac-
therized by a critical micelle concentration (CMC), which
depends on the properties of the native copolymer. CMC is
strictly related to micelle disassembly in unimers upon dilu-
tion [35]. Thus, low CMC values are expected to give nano-
systems unsensitive to dilution and thus stable after i.v. in-
jection. In general, micelles are smaller than corresponding
NPs (10-100 nm versus > 100 nm) and unimers in the mi-
celles are in a dynamic equilibrium with unimers in the bulk.
A clear distinction between micelles and NPs is not always
possible with PEGylated polyesters as demonstrated for
PEG-PDLLA [34]. Thus, micelles are sometimes referred to
as NPs. Aggregation behavior strongly depends on copoly-
mer composition/architecture. As far as the molecular weight
of the hydrophobic block increases, hydrophobic core be-
comes more solid-like as it happens for NPs whereas smaller
PDLLA blocks produces micelle-like assemblies. Neverthe-
less, diblock copolymers can form also bilayered vesicles
resembling liposomes and named polymerosomes. Similarly
to nanocapsules, polymersomes are indeed vesicular systems
in which th e drug is confined to a reservoir or within a cavity
surrounded by a polymer coating. Accurate selection of
polymer molecular weight, hydrophilic/hydrophobic ratio
and chemistry impart polymersomes with a broad and tun-
able range of carrier properties. These systems are capable of
encapsulating a large range of therapeutically-active water-
soluble molecules and biomolecules, with considerable work
being done to engineer the release of those encapsulants at
the desired place and time [31, 36, 37].
A specific nanosized carrier can be obtained by playing
on block copolymer composition, and method of preparation.
Depending on hydrophilic/hydrophobic balance, different
solubility profiles in water or organic solvents occur, thus
dictating the preparation method and the expected type of
nanostructure [34]. Over the well-known conventional meth-
ods to prepare NPs, such as solvent diffusion method and
formation on nano-emulsion templates [26, 38], numerous
examples of original methods to form NPs entrapping lipo-
philic and hydrophilic molecules are available [39, 40]. For
example, we developed a Melting Sonication technique
which, exploiting the intrinsic solubility and the low melting
temperature of PCL-PEG block copolymers, does not make
use of organic solvents and gives core-shell NCs with high
PEG surface coverage [41-43]. Micelles and polymersomes
organize spontaneously by direct dissolution in water or un-
der dyalisis from copolymer solutions in dimethylformam-
mide, tetraydrofurane, N-methylpyrrolidone. Hydration of a
polymer film under sonication is another method proposed to
form self-assemblies [34].
For each specific preparation technique, drug encapsula-
tion efficiency is again controlled by the overall properties of
the polymer. It has been observed that in the case of micelles
the longer is the PLA chain length, the greater is the loading
of hydrophobic drugs [44]. Nevertheless, chain lenght con-
trol overall size of the system. As recently reviewed [21],
many factors may influence drug release from PEG–PLA
based nanocarriers, that is molecular weight, chain length of
PEG or PLA, and PEG/PLA ratio in the polymer .
To remain well dispersed in a liquid, PEGylated NCs,
like all types of colloids, need to be stabilized using am-
phiphilic molecules or colloid protecting agents. Indeed, the
large surface area of NCs creates high total surface energy,
which is thermodynamically unfavorable. Furthermore, the
systems can be destabilized along time due to different
physical and chemical phenomena, such as the degradation
rate of the base materials [45]. In this context, the most
commonly used technique to improve the physical and
chemical stability of NCs is freeze-drying the colloidal dis-
persion, often making use of proper cryoprotectants. By this
way, powders for intravenous injection that are stable for
long time and can be easily redispersed in water before the
administration can be achieved [46]. In the case of PEGy-
lated NCs, the stability of the system is generally increased,
since the external hydrophilic fringe of PEG acts as steric
stabilizer as well as collapse temperature modifier, thus pro-
tecting the product against freezing and drying stresses.
Nevertheless, it has been found that the success of freeze-
Fig. (1). PEGylated polyester-based nanocarriers: A) main architectures of PEGylated polyesters; B) nanostructures formed by PEG-
polyesters assembly.

Pegylated Polyester-Based Na noncologicals Current Topics in Medicinal Chemistry, 2014, Vol. 14, No. 9 5
drying of PEGylated polyester-based NCs depends on differ-
ent PEG properties, including the molecular weight, the con-
centration and the location at NC surface of PEG chains. It
was demonstrated, in fact, that the covalent attachment of
PEG to NC surface can induce the PEG crystallization with
the formation of intra- and interparticular bridges during
freezing, thus resulting in aggregated particles after water
removal. In light of this observation, NC dispersion in an
amorphous matrix acting as a crioprotectant (e.g., threalose,
sucrose) during freezing may maintain the PEG chains in a
pseudo-hydrated state, thus avoiding NC aggregation phe-
nomena [47, 48].
3. INJECTABLE PEGYLATED POLYESTER-BAS ED
NANONCOLOGICALS
3.1. Biologically-Oriented Design Rules
The main reason that prompts the increasing interest in
nanomedicines for cancer therapy relies in the possibility to
modify and improve pharmacokinetics and sometimes phar-
macodynamics of anticancer drugs, thus ameliorating the
therapeutic response and efficacy. However, the complexity
of in vivo biological systems poses multiple barriers (Fig. 2)
that severely limit the access of a nanomedicine to its target
and have to be overcome to fully exploit the theoretical po-
tential of nanoncologicals [8, 49, 50]. The major challenges
for intravenous nanoncologicals include escape from clear-
ance mechanisms in the body, increasing the dose fraction
reaching tumor, maintainance of high drug levels in tumor
tissue and inside cells, as well as promoting endosomal es-
cape, that is especially important for nucleic acid fragments.
Natural elimination processes include both renal clear-
ance and MPS uptake. A huge number of studies have re-
ported that composition, size distribution and surface proper-
ties are the most important parameters influencing the ulti-
mate fate of NCs in the body. Thus, a fine control of NC
architecture has to be considered to ensure prolonged blood
circulation as well as to increase the probability to reach the
diseased tissues. It is well known that filtration of particles
through the glomerular capillary wall is highly dependent on
molecule size and is referred to as the filtration-size thresh-
old. Molecules with a diameter less than 6 nm are typically
filtered, while those more than 8 nm are not typically capa-
ble of glomerular filtration [51]. Concerning MPS uptake,
following intravenous administration, NCs tend to adsorb
plasma components such as opsonins, including immuno-
globulins, apolipoproteins, components of complement sys-
tem, and clotting factors onto their surface. Opsonins medi-
ate NC recognition by monocytes and subsets of tissue
macrophages and opsonized NCs tend to be rapidly cleared
from the circulation by phagocytic cells of MPS and accu-
mulated in the liver (Kuppfer cells) [52, 53]. Thus, the liver
acts as a reservoir toward NCs conditioning their rapid first-
phase disappearance from the blood and, in case of biode-
Fig. (2). The multiple biological barriers encountered by PEGylated nanocarriers upon intravenous injection.

6 Current Topics in Medicinal Chemistry, 2014, Vo l. 14, No. 9 Conte et al.
gradable systems, their second-phase release in the body
under degraded and excretable form. This biodistribution can
be of benefit for the chemotherapeutic treatment of MPS
localized tumors (e.g. hepatocarcinoma or hepatic metastasis,
bronchopulmonary tumors, myeloma and leukemia) but
rather undesirable in case of different tumor location. Ide-
ally, an injectable NCs has to be smaller than 100 nm to
avoid internalization by the MPS. Furthermore, particles
with hydrophobic surface or positive charge show a high
affinity to opsonins [13, 53, 54].
Coating NC surface with hydrophilic polymers is the
most accreditated and effective method to make them “in-
visible” to the immune system, creating long-circulating
NCs, known also as “stealth”. Although several hydrophilic
polymers have been tested to give biomimetic carriers, PEG
remains the gold standard to provide biomimetic properties
[18, 55]. In this context, the modification of NC surface with
PEG is the most useful strategy to prolong the longevity of
NCs in the blood circulation as well as their passive accumu-
lation in the tumor sites. In th is way, in fact, it is possible to
create a hydrated water barrier that provides good steric hin-
drance to the attack of phagocytes [56, 57]. Moreover, a
steric stabilization limits also aggregation between particles
themselves in the blood and contributes to system stability in
biological environments. Gref et al [58] were the first to re-
port the advantages of PEGylation on PLGA-PEG NPs, re-
sulting in a substantial increase in blood residence time.
The properties of PEG corona, including thickness, chain
molecular weight, PEG surface density and conformation are
considered as critical factors to achieve stealth characteris-
tics. Particles with covalently bound PEG chains exhibit
longer blood circulation half-lives than similar particles with
only surface adsorbed PEG whereas a PEG of 2000 Da or
greater is required to achieve increased MPS-escape, due in
part to the increased chain flexibility [13, 53, 58]. Concern-
ing the shell conformation, it is widely recognized that at
low surface coverage, PEG chains have a larger range of
motion and will typically take on what is termed a “mush-
room” configuration, where on average they will be located
closer to the surface of the particle. Very low surface cover-
age can also lead to gaps in the PEG protective layer, where
opsonins can freely bind to the NC surface. On the other
hand, at high surface coverage the PEG chains range of mo-
tion will be greatly restricted and they will most often exhibit
a semi-linear or “brush” configuration. Although a high sur-
face coverage ensures that the entire surface of NC is cov-
ered, this method also decreases the mobility of the PEG
chains and thus decreases the steric hindrance properties of
the PEG layer. Interestingly, a threshold of 1-2 nm space
between the PEG chains was estimated for minimal protein
absorption [58].
Despite extensive research devoted to the design of in-
jectable PEGylated NCs, and the clinical approval of several
PEGylated nanomedicines, some recent studies report that
repeated administrations of PEGylated liposomes [59-61] or
polymeric NPs [62, 63] at specific intervals can induce the
unexpected accelerated blood clearance (ABC) phenomenon.
The mechanism of ABC is still not fully understood, but it
has been suggested that the formation of anti-PEG IgM anti-
bodies by the spleen occurs upon the first injection; the IgM
binds to the PEG of the second dose and activates the com-
plement system, thereby leading to opsonization with C3
fragments of PEG and an enhanced uptake by Kupffer cells
[18, 64]. To date, the magnitude of this event is not com-
pletely clear, despite it was reported that ABC is strictly cor-
related to several parameters, such as PEG-surface density,
PEG-chain length, time intervals between injections, N P
doses and properties [65]. Therefore, the development of a
strategy to attenuate and/or abrogate the immunogenicity of
PEG-coated NCs without significantly compromising their in
vivo performance would be highly desirable for the further
development of novel drug delivery systems.
Further decoration of NC surface with targeting ligands
overexpressed on cancer blood vessels or cancer cells can be
useful to improve specificity of stealth NCs. Nevertheless,
after leaving the highly leaky tumor vasculature, there are
quite a number of anatomical and physiological barriers that
a targeted NC needs to overcome before reaching cancer
cells. These include the presence of pericyte-, smooth muscle
cell- and fibroblast-based cell layers between endothelial and
tumor cells, the high cellular density within solid malignan-
cies, and the high interstitial fluid pressure that is typical of
tumors. Therefore, and also because of the binding-site bar-
rier, which further limits the penetration of actively targeted
nanomedicines into the tumor interstitium, actively targeted
nanomedicines tend to have problems finding their target
cells, and they can fail to demonstrate an advan tage over
passively targeted formulations [66-68].
3.2. Targeting Cancer: Strategies and Applications
In order to obtain an optimized therapeutic response, the
modulation of NC properties as well as their journey from
the site of administration to the site of action is a crucial
point to consider. The ideal and desired effect of an antican-
cer nanomed icine, in fact, is that the whole administered
dose reaches the target site, thus affecting as few as possible
the healthy tissues. On this basis, during the last few years, a
wide range of polyester-based NPs have been developed
through the application of different targeting strategies (Fig.
3) with the aim to promote the accumulation of one or more
anticancer agents inside tumors in general and specifically
inside cancer cells.
4. PEG-POLYESTER NANOCARRIERS FOR PAS-
SIVE TARGETING
The simplest strategy that allows the preferential accu-
mulation of an intravenous nanomedicine in solid tumors
relies on passively targeting mechanisms, thus exploiting the
abnormal properties of tumor vasculature through EPR ef-
fect. The impaired lymphatic drainage and the pathophysi-
ologic features of tumor blood vessel, in fact, enables mac-
romolecules to extravasate into extra vascular spaces and
accumulate inside tumour tissues [9]. Passively targeted PE-
Gylated NPs remains entrapped mainly in tumor interstitium,
where they deliver drug to cancer cells. Indeed, PEG confers
a hydrophilic surface to NPs that strongly limit their uptake
inside cells. The consequent formation of stable drug cues
inside the tumor can contribute to drug penetration in the
farthest area of the tumor, where hypoxic conditions are es-
tablished, and to limit drug resistance [69]. To this purpose,

Pegylated Polyester-Based Na noncologicals Current Topics in Medicinal Chemistry, 2014, Vol. 14, No. 9 7
several types of PEG-polyester NCs were developed and
investigated for their potential in different tumor pathologies.
In particular, among the most of 450 systems designed in the
last 20 years, only 30 of them have been tested in preclinical
phase [66]. It is worthy to note that, as evidenced in Table 2,
only three PEG-polyester systems can be considered real
nanoncologicals already approved in the clinic (Genexol®-
PM) or undergoing clinical trials (Nanoxel-PM™ and DTX-
PNP).
Genexol®-PM represents the first example of marketed mi-
celles based on PEGylated polyesters. It is a sterile, lyophi-
lized paclitaxel micellar formulation based on a diblock
PEG-PDLLA. It was launched in the Korean Market by the
Samyang Group in February 2007 for the treatment of breast
cancer and advanced non-small cell lung cancer. It is now
undergoing phase III clinical trials for the treatment of recur-
rent or metastatic breast cancer, a phase I/II trial in combina-
tion with carboplatin as a firstline treatment of advanced
ovarian cancer, a phase II trial in combination with cisplatin
in locally advanced head and neck cancer and finally a phase
II trial in combination with doxorubicin in advanced breast
cancer [70, 71]. Meanwhile, docetaxel-loaded mPEG-
PDLLA micellar formulation (Nanoxel-PM™) has entered
Phase I clinical trial to evaluate bioequivalence with
Taxotere® (the conventional docetaxel formulation) in a
multi-center, open-label, randomized, crossover study. In
fact, preclinical pharmacokinetic study in mice, rats and bea-
gle dogs revealed that this formulation exhibited similar
pharmacok inetic profiles compared to Taxotere® [72], thus
suggesting a possible micelle disassembly in the blood-
stream, as evidenced by previous findings on similar mi-
celles [73]. Finally, DTX-PNP is a formulation of polymeric
NPs developed by Samyang comprising a mixture of mono-
valent metal salts of PLA, amphiphilic diblock copolymers
of PEG-PLA and docetaxel. Also in this case, the antitu-
moral activity of the resulting micelles in mice models was
similar to that of free drug, despite micelle capacity to in-
crease radiotherapy efficacy in mice bearing A549-derived
tumors [74]. A Phase I clinical trial of this formulation for
the treatment of various solid malignancies is currently un-
derway in Korea to determine the Maximum Tolerated Dose
and evaluate the safety and pharmacokinetics [75].
Concerning PEGylated PLGA, recent studies demon-
strated that correspondent NPs are able to ameliorate biodis-
tribution of the anticancer drug in mice thus increasing its
accumulation at tumor level, where it is eviden t a double
effect: great NC internalization in cells and/or release of the
drug from the carrier in the extracellular matrix, both de-
pending on the properties of PEG segment and the hydro-
philic/hydrophobic balance of the copolymer. Overall these
phenomena caused a stronger antitumor effect compared the
free drugs [76-78]. A physiologically based pharmacokinetic
model was recently developed and evaluated to simulate the
mass-time tissue distribution profiles of five NP formula-
tions administered by intravenous injection in mice [79].
NPs were previously developed from different PEG-PLGA
copolymers with varied ratios between lactic acid, glycolic
acid and ethyleneoxide units [80]. Based on the pharmacoki-
netic model, the characterized physicochemical properties of
a novel formulation were used to predict the biodistribution
profile and found close to experimental data, thus suggesting
that property-biodistribution relationships are adequately
modelled.
It is now well established that structure (nanosphere, po-
lymersome, micelle) and physical-chemical properties of the
base material (CMC, solubility, crystallinity) can have a
strong impact on in vivo stability and biological fate of PE-
Gylated NCs [42, 81]. For instance, it was demonstrated that
PEG-PDLLA micelles disassemble rapidly in the blood-
stream following intravenous administration in mice [73,
82], which could explain the similar activity profile of clini-
cal approved PEG-PDLLA micelles to that of free drug [72].
Nevertheless, the increase of the molecular weight of
PDLLA block in the copolymer may have detrimental effects
on micelle stability. Contrariwise, the longer the PEG chain
length, the more stable is the resulting nanostructured carrier
[83].
Fig. (3). Main strategies to specifically target cancer cells/tissue with PEGylated nanocarriers: targeting tumor cells (A); targeting tumor
vasculature (B); physical targeting through stimuli-sensitive materials (C).

8 Current Topics in Medicinal Chemistry, 2014, Vo l. 14, No. 9 Conte et al.
Table 2. PEGylated polyester-based nanoparticles currently under preclinical/clinical investigation or in the market.
Polymer
(Mw or Mn)
Size
Zeta Potential Drug
Targeting
protein/
receptor
Indication Administra-
tion route
Stage of devel-
opment Ref
PEG shell
mPEG-PDLLA
(Mw= 2000-1750)
20/50 nm
NR Paclitaxel -
Breast cancer, Ad-
vanced non-small cell
lung cancer
Radiosensitizer for
non-small cell lung
cancer
Intravenous
Intravenous
Approved/Phase
I/ Phase II/Phase
III (Genexol®-
PM)
Preclinical
[70, 71]
mPEG-PDLLA
(Mw= 2000-1765) NR Docetaxel - Breast cancer Intravenous
Phase I
(Nanoxel-
PM™)
[72]
mPEG-PLA
(Mw= 2000-5000)
10/50 nm
NR Docetaxel -
Various solid malig-
nancies Intravenous Phase I (DTX-
PNP) [75]
mPEG–PLA
(Mn= 2000-1800)
35 nm
-2.9 mV Sirolimus - Lung cancer Intravenous Preclinical [167]
mPEG-PLA-b-
Polyargin-
ine(R15)
(Mn= 2000-3000)
54 nm
+34.8 mV EGFR-siRNA - Breast cancer Intravenous Preclinical [95]
PEG-PLA 170/200 nm
+13.8 mV siRNA -
Hepatic and breast
cancer Intravenous Preclinical [94]
TPGS-PLGA
(Mn= 1000-25000)
90/140 nm
-11.0/-21.0 mV Docetaxel - Breast cancer Intravenous Preclinical [78]
PEG-PLGA
(Mn=29300)
PEG-PCL
(Mn= 22400)
112/190 nm
+7.7 mV Paclitaxel - TLT tumor Intravenous Preclinical [77]
(PGA-co-PCL)-
TPGS
(Mw= 20235)
200/260 nm
-31.2 mV Paclitaxel - Lung cancer Intratumoral Preclinical [168]
mPEG-PCL
(NR) NR Paclitaxel and
berbamine - Gastric cancer Intratumoral Preclinical [91]
mPEG–PCL
(Mw=4000-20000)
70 nm
NR Docetaxel - Hepatic cancer Intravenous Preclinical [81]
PEG–PCL
(NR)
100/250 nm
NR Paclitaxel - Pancreatic cancer Intravenous Preclinical [88]
PCL–PEG–PCL
(NR)
100 nm
NR Paclitaxel - Breast cancer Intravenous Preclinical [89]
PEO-PCL
(Mw= 2000-4200)
PEO-PCL-PEO
(Mw= 2000-8000-
2000)
60/100 nm
-10.9/-18.3 mV Docetaxel - Breast cancer Intravenous Preclinical [41]
PEG–PCL
(Mw= 2000-4000)
25 nm
NR Doxorubicin - Colon cancer Intravenous Preclinical [90]
PEG-PAsp(DET)-
PCL
(PEG Mn= 5000)
70/100 nm
+7.0/+10.0 mV
Rapamycin
and siRNA
targeting Y-
box binding
protein-1
- Prostate cancer Intravenous Preclinical [96]

Pegylated Polyester-Based Na noncologicals Current Topics in Medicinal Chemistry, 2014, Vol. 14, No. 9 9
(Table 2) contd….
Polymer
(Mw or Mn)
Size
Zeta Potential Drug
Targeting
protein/
receptor
Indication Administra-
tion route
Stage of devel-
opment Ref
PEG-PCL
(Mn=5000-10000)
80/90 nm
NR
Paclitaxel,
cyclopamine
and gossypol
- Ovarian cancer Intraperitoneal Preclinical [92]
mPEG-PCL
(Mw=
7000/17000)
30/56 nm
-4.9/-10.8 mV Paclitaxel - Biodistribution in
healthy mice Intravenous Preclinical [86]
mPEG-PCL
(NR)
38 nm
NR Paclitaxel - Pulmonary carcinoma Intravenous Preclinical [87]
Targeted PEG shell
PSMA ligand-
PEG-PLGA
(Mw= 5000-
16000)
NR Docetaxel PSMA
Non-small cell lung
cancer and prostate
cancer
Intravenous Phase II
(BIND-014) [101]
NR7 peptide-
PEG-PLGA
(Mw= 5000-
28000)
135 nm
-13.0 mV Doxorubicin EGFr Ovarian cancer Intravenous Preclinical [102]
FA-PEG- PLGA-
DOX (Mw = 3400-
8000)
105/112 nm
NR Doxorubicin FAr Epidermal carcinoma Intravenous Preclinical [110]
TPGS –PLGA-
CA
(Mw = 1000-
34000)
100/140 nm
-13.9/-23.6 mV Docetaxel NR Cervical cancer Intratumoral Preclinical [112]
LyP1- PEG-
PLGA
(Mw = 3500-
34000)
90 nm
-10.4 mV Model drug Neuropilin-
1/Integrin
Metastasis lymph
nodes Intravenous preclinical [113]
AS1411-PEG-
PLGA
(Mw = 3500-
15000)
154 nm
-32.9 mV Paclitaxel Nucleolin Glioblastoma Intravenous preclinical [107]
mPEG-PLGA-
PLL-EGF
(Mw= 42300)
190 nm
+1.9 mV Cisplatin EGFr Ovarian cancer Intravenous Preclinical [106]
mPEG-PLGA-
PLL-cRGD
(Mw= 11000)
164 nm
+2.77 mV Bufalin Integrin Colon cancer Intravenous Preclinical [105]
mPEG-PLGA-
PLL-cRGD
(Mw= 11000)
180 nm
+0.6 mV Mitoxantrone Integrin Breast cancer Intravenous Preclinical [104]
HA-PEG-PCL
(Mw= NR)
95 nm
NR Doxorubicin HAr Ehrlich ascites tumor Intravenous Preclinical [111]
FA-PEG-PCL
(Mw=5100-2900)
44 nm
NR SPION FAr Hepatic cancer Intravenous Preclinical [109]
EGF-PEG–PCL
(Mw= 3500-1200)
60 nm
NR - EGFr Breast cancer Intravenous Preclinical [103]
GMT8- PEG-
PCL
(Mw = 3000-
15000)
100/113 nm
-3.4 mV Docetaxel Receptor on
U87 cells Glioblastoma Intravenous Preclinical [108]

10 Current Topics in Medicinal Chemis try, 2014, Vo l. 14, No. 9 Conte et al.
(Table 2) contd….
Polymer
(Mw or Mn)
Size
Zeta Potential Drug
Targeting
protein/
receptor
Indication Administra-
tion route
Stage of devel-
opment Ref
Stimuli-responsive P EG shell
pH-respo nsive
H7K(R2)2- PEG-
PLGA
(Mn= 6000-3000)
46/54 nm
0.8/+0.5 mV Paclitaxel
H7K(R2)2 (pH
sensitive pep-
tide)
Breast cancer Intravenous Preclinical [125]
mPEG–Pep-PCL
(Mw= 13000)
86 nm
NR Docetaxel MMP2/9 Hepatic cancer Intravenous Preclinical [128]
ALMWP -PEG-
PCL
(Mn= 3000-20000)
107/134 nm
-23.3/+18.4
mV
Paclitaxel
MMP-2/9
activatable
protamine
Glioblastoma multi-
forme Intravenous Preclinical [127]
pH- and thermo- responsive
mPEG-P(HPMA-
Lac-co-Hys):
mPEG-PLA
(NR)
150 nm
NR Rapamycin - Colon cancer Intravenous Preclinical [126]
P(TEGMA-co-
NHSMA)-b-PCL
(Mn= 35000-4500)
52/70 nm
NR
Doxorubicin
- Melanoma Intravenous Preclinical [124]
Light- sensitive
mPEG- PLGA
(NR) NR Temoporfin - Breast and lung cancer Intravenous Preclinical [76]
mPEG-PCL
(Mw= 2000-4200)
60/100 nm
-7.9/-13.8 mV
Docetaxel and
Zinc(II)-
phthalocyanine
- Breast cancer Intravenous Preclinical [42]
PEGylated theranostic
mPEG–PLA
(Mw= 2000-
20000)
50/70 nm
-16.7/+17.9
mV
plasmid pDNA
SPIONs - Biodistribution in
healthy mice
Intravenous/
intraperitoneal Preclinical [134]
PLGA
(Mw= 7000-
17000)
mPEG-PLGA
(Mw= 4600-
10040)
mPEG-PCL
(Mw= 5000-
13100)
240 nm
-2.0 mV
Paclitaxel
Doxorubicin
SPION
-
Cancer therapy and
magnetic resonance
imaging in colon carci-
noma
Intravenous Preclinical [169]
mPEG–
PLGA/EPL
(NR)
85/105 nm
+29.1/+31.1mV
Doxorubicin
Paclitaxel
Survivin
siRNA
BSA–Au
- Melanoma Intravenous Preclinical [135]
NR= not reported.
Abbreviations: ALMWP = activable low molecular weight protamine; CA = cholic acid; cRGD = cyclic arginine-glycine-aspartic acid; DOX = doxorubicin; EGFR = epidermal
growth factor receptor; EPL =  -polylysine; FA(r) = folic acid (receptor); EGF(r) = human epidermal growth factor (receptor); HA(r) = hyaluronic acid (receptor); HPMA = N-(2-
hydroxypropyl)methacrylamide; NHSMA = N-hydroxysuccinimide methacrylate; PAsp(DET) = poly{2-[(2-aminoethyl)amino] ethyl aspartamide}; PCL = poly(-caprolactone);
pDNA = plasmid DNA; mPEG = methoxy-PEG; Pep = Gelatinase cleavable peptide; PLA = poly(lactic acid); PGA= poly(glycolide); PLGA = poly(lactide-co-glycolide); PLL=
poly(lysine); PSMA = Prostate specific membrane antigen; P(TEGMA)= methoxytri(ethyleneglycol) methacrylate; siRNA = small interfering RNA; SPION = superparamagnetic iron
oxide nanoparticles; TPGS = d-alpha tocopheryl polyethylene glycol; VEGF = vascular endothelial growth factor.

Pegylated Polyester-Based Na noncologicals Current Topics in Medicinal Chemistry, 2014, Vol. 14, No. 9 11
Poor stability of PEG-PDLLA micelles has prompted to
the use of PEG-PCL polymers. In fact, PEG-PCL micelles
with a semicrystalline and more hydrophobic core than PEG-
PDLLA micelles are likely to have greater kinetic stability
and slower rate of disassembly [84]. Also in this case, hy-
drophilic/hydrophobic ratio is a critical parameter for stabil-
ity, since fluorogenic micelles based on PEG45-PCL21 (Mn =
6960 Da, PEG Mn around 2000 Da) are rapidly disrupted
and dissociated in the presence of serum [85]. Despite the
different in vitro stability of NPs prepared from PEG-PCL of
different molecular weight, the systems showed a similar in
vivo pharmacokinetic profile of the delivered drug in mice
[86]. On the other hand, a strong accumulation of the system
in different cancer organs through passive targeting mecha-
nisms was found by others. Biodistribution studies carried
out on mice models showed an increased accumulation of
PEGylated PCL micelles/NPs delivering taxanes in solid
tumors, which determined a decrease in tumor growth inhibi-
tion [81, 87, 88]. This effect was explained also in term of
facilitated penetration of the drug inside tumor [81]. At the
same time, core shell paclitaxel-loaded micelles based on
triblock PCL-PEG-PCL copolymers evidenced a similar
anticancer effect of the free drug in vivo, while survival was
significantly improved [89]. Also copolymer architectures
(diblock, triblock or star-shaped) can influence therapeu tic
outcome. Gao et al [90], for example, demonstrated that core
shell micelles based on star shaped PCL-PEG block copoly-
mers showed a more significant inhibitory effect on tumor
growth than free doxorubicin likely due to higher apoptosis.
Regardless of copolymer composition, superior anticancer
effects can be reached through the entrapment of two or
more anticancer compounds with different mechanisms in
the same passively targeted NPs [91, 92].
The molecular weight of the copolymer plays a key role
not only on the stability and on the in vivo fate of the carrier,
but also affects interaction with cells and drug accumulation.
It was recently reported, in fact, that the employment of
low molecular weight PEG-PCL diblock copolymer (5 or 19
PCL units linked to PEG 750 or 5000 Da, respectively) can
enhance the cytotoxic potential of paclitaxel-loaded micelles
through the inhibition of the P-glycoprotein (P-gp), which is
overexpressed in many kind of cancer cells and responsible
of the MDR [93].
From a preclinical point of view, other types of NPs for
siRNA delivery based on PEG-PLA block copolymers modi-
fied with cationic elements are now under investigation in
different cancer models. Both cationic lipids, such as N,N-
bis(2-hydroxyethyl)-N-methyl-N-(2-cholesteryloxycarbonyl
aminoethyl) ammonium bromide (BHEM-cholesterol) [94],
and cationic polymers, such as poly(amino acids) [95, 96],
have been tested to the purpose.
5. ACTIV ELY-TAR GETED PEG-POLYESTER
NANOCARRIERS
The most common approach to improve specifivity of
passively targeted nanomedicines remains decorating NC
surface with different functional and targeting motifs, thus
building an actively-targeted nanomedicine with a higher
specificity to the target site [97]. Research has focused on
several targeting ligands, including antibodies, aptamers,
peptides, small molecules and ligands binding particular re-
ceptors overexpressed in cancer cells or blood vessels [6, 23,
98, 99]. In this way, an amplified and enhanced pharmacol-
ogical effect and/or a more remarkable cellular uptake of the
nanomedicine was expected. Efficient binding and internali-
zation requires that receptors are expressed exclusively on
target cancer sites relative to normal cells. Up to now, only
antibody-based targeted nanomedicines have been approved
for clinical use in spite of significant advances made at pre-
clinical level. Nevertheless, several targeted delivery systems
are under clinical trials [24], such as transferrin receptor tar-
geted cytotoxic platinum-based oxalip latin loaded in
liposomes (MBP-426), transferrin receptor targeted cyclo-
dextrin-containing NPs with siRNA payload (CALAA-01),
and prostate-specific membrane antigen (PSMA) targeted
polymeric NPs containing DTX (BIND-014). BIND-014
(BIND Therapeutics, Cambridge, MA, USA) is the first ex-
ample of targeted polymeric NPs of PEG-PLGA. Targeting
to PSMA, which is abundantly expressed on the surface of
cancer cells and new blood vessels of solid tumors, results in
a DTX delivery up to 20 times higher than an equivalent
dose of Taxotere in preclinical cancer models [100]. BIND-
014 is now undergoing in a phase II clinical trial, for the
treatment of non-small cell lung cancer and prostate cancer,
to determine the maximum tolerated dose and assess its
dose-limiting toxicities [101].
In Table 2 several examples of actively targeted nanoon-
cologicals based on PEGylated polyesters under preclinical
studies are reported. For the majority of cases, the targeting
strategy implies the chemical functionalization of the base
copolymers with the targeting ligand in order to assure the
exposition of these functional moieties on NC surface. As
previously explained, the targeting ligand can be a peptide
[102-106], an aptamer [107, 108] or a small molecule [109,
110]. Through these motifs, PEGylated NCs are able to se-
lectively interact with particular receptors overexpressed in
many cancer cells, both of solid tumors [103, 104, 106, 109-
112] and lymphatic metastasis [113] (cellular targeting), or
in endothelial cells of angiogenic blood vessels [104, 105]
(vascular targeting).
It is worthy to note that two or more targeting ligands can
be applied in the same carrier in order to achieve a multi-
functional and stronger outcome. Recently, small PEG-PCL
micelles functionalized with folic acid (FA) have been de-
veloped and loaded with super paramagnetic iron oxide.
Long time-circulation in the blood, extensive cell uptake and
a strong suitability in MRI diagnosis was demonstrated, thus
pointing at their use as a probe for tumors overexpressing
folate receptor [109].
An important drawback of targeted NPs is that they can
paradoxically lose targeting ability in a biological environment
[114]. Indeed, interaction with other proteins in the medium
and the formation of a “protein corona” can screen the target-
ing molecules on the surface of NPs [115] while the so called
"binding site barrier" effect may confine the drug in the peri-
vascular regions retarding drug/NP tumor penetration [116].
On the other hand, the extent of targeting ligand presentation
at NP surface can be much lower than theoretical, which can
result in poor benefits of the targeting approach [117].

12 Current Topics in Medicinal Chemis try, 2014, Vo l. 14, No. 9 Conte et al.
6. STIMULI-SENSITIVE PEG-POLYESTER
NANOPARTICLES
Drug release from a nanomedicine at tumor level can be
achieved through endogenous or external stimuli with the
aim to improve anticancer drugs specificity. The local
changes that occur in solid tumors can be exploited to trigger
drug release at extracellular level (decreased pH in tumor
interstium, increased temperature, presence of specific en-
zymes), intracellular level (decreased pH and reducing envi-
ronment of endolysosomes), or both. Furthermore, external
stimuli such as temperature increase, application of ultra-
sound/light/magnetic field can not only activate drug inside
tumors but also drive NCs to the diseased tissue, as in the
case of magnetic field.
NCs made up of pH- or thermo- sensitive components,
including block copolymers, disintegrate under conditions of
increased temperature or decreased pH values and release
their cargo in the pathological site. To this respect, several
smart materials have been developed so far and revised in
several recent reviews [118-120]. Furthermore, NC targeting
inside cancer cells can be improved by generating a variation
of NC surface properties at sites of disease, an approach in-
dicated as “shedding” [121]. Nevertheless, for anticancer
drugs with an intracellular target site and needing escape
from the lysosomes before exhibiting biological effects,
clever incorporation of functional groups on NC surface or
use of amphiphilic block copolymers with exquisite sensitiv-
ity can be of great potential.
Stimuli-responsive polymeric micelles based on block
copolymers have been developed extensively by Bae et al.
[97]. Indeed, mixed micelles formed with conventional PEG-
PLA and pH sensitive copolymers (poly(L-histidine)-b-
polyethylene) gave NPs that dissolved at a pH value that can
be tuned by changing the ratio of copolymers, leading to pH-
targeted drug release [122]. Improved accumulation of mi-
celles was also obtained by decorating NPs with a TAT-
peptide that promote tumour cell-specific internalization
[123]. More recently, several dual pH- and temperature-
responsive block copolymers containing a PCL hydrophobic
block with a poly(triethylene glycol) block were copolymer-
ized with an amino acid-functionalized monomer and em-
ployed to form micelles in aqueous solution[124]. These
micelles were investigated for the delivery of doxorubicin in
an MDA-MB-435 (melanoma) xenograft subcutaneous tu-
mor model. A faster drug release in tumor acidic environ-
ments as well as improved anti-tumor efficacy compared to
free doxorubicin and lower associated systemic toxicity was
observed. Tumor-specific pH-responsive peptide-modified
polymeric micelles based on PEG-PLGA block copolymers
containing paclitaxel were also developed . The in vivo tar-
geting activity on tumor/endothelial cells and the in vivo
anti-tumor activity of these micelles were demonstrated in
MCF-7 tumor-bearing mice, where they induced a remark-
able anti-tumor and anti-angiogenic effects [125]. Finally,
dual-responsive mixed micelles for colon delivery of ra-
pamycin have been recently prepared from PEG-b-poly(N-
(2-hydroxypropyl) methacrylamide dilactate)-co-(N-(2-
hydroxypropyl) methacrylamide-co-histidine)), that is
mPEG-P(HPMA-Lac-co-His), and mPEG-PLA [126]. Mi-
celles demonstrated a remarkable anticancer activity in tu-
mor mouse models. Finally, PEGylated PCL NPs for the
delivery of taxanes were developed through the modification
of the PEG-PCL with protamine to achieve a matrix metal-
loprotease-sensitive system [127] or with a gelatinase-
cleaved peptide [128], in order to exploit the increased pro-
tease expression at the tumor sites.
As stated above, the selectivity of PEGylated polyester
NPs toward cancerous cells can be increased also through ex-
ternal stimuli. This is the case of light-activated nanongologi-
cals entrapping a photosensitizer (PS), which can be activated
at cancer cell level through light of an appropriate wavelength.
With this idea in mind, temoporfirin-loaded PEG-PLGA NPs
were recently developed and tested for their effects on cancer
cell lines as well as for the capability to accumulate in vivo at
cancer level [76]. Of note, in vivo imaging performed on
athymic nude-Foxn1 mice at 96 h post-injection revealed a
higher tumor-to-skin ratio for PEGylated NPs as compared to
both their non-PEGylated counterpart and free temoporfirin,
likely resulting in limited side effects of treatment. More re-
cently, light-activated PEG-PCL NPs entrapping a PS in com-
bination with docetaxel, a conventional anticancer drug, dem-
onstrated superior ability to kill cancer cells both in vitro and
in an orthotopic mice model of melanoma [42]. The contem-
porary improvement of animal survival as compared to mice
treated with free anticancer drug suggest a potential reduction
of side effects in healthy tissues.
7. PEG-POLYESTER NANOCARRIERS AS THER-
ANOSTICS AND IMAGING AGENTS
NC surface can be engineered not only with targeting
motifs, but also with a wide range of functional groups, thus
allowing the design of multifunctional nanostructures. Ther-
anostic nanoncologicals can combine, besides a specific tar-
geting agent, a cell-penetrating agent, a stimulus-sensitive
element to trigger drug release, a stabilizing coating to en-
sure biocompatibility and two or more therapeutic com-
pounds, also an image probe (such as Quantum Dots, gold,
iron oxide, a radioactive agent). This approach can be used
for simultaneous imaging, diagnosis and treatment of tu-
mors. Several recent reviews have discussed engineering
strategies, physiochemical characteristics and biomedical
applications of multifunctional NCs [22, 129-133].
Recently, chitosan and polyethyleneimine (PEI) coated
magnetic micelles based on a block copolymer of mPEG-
PLA have been proposed for dual-purpose magnetic reso-
nance imaging and gene therapy [134]. mPEG-PLA micelles
contained magnetite in the core whereas chitosan, PEI and a
plasmid DNA were embedded in the shell. The resulting
micelles display high MRI relaxivity, can efficiently trans-
fect various cell lines, such as HEK293, 3T3 and PC3 cells
in vitro and deliver genes with high transfection efficiency.
The biodistribution experiments in mice showed that this
system accumulate in the liver, lung, spleen and prostate
after a single intravenous injection, whereas in-vivo clear-
ance studies further demonstrated that these particles are
biocompatible and safe. As previously described, small
PEG-PCL micelles functionalized with FA were developed
and tested in MRI diagnosis for tumor detection [109].
Another example of a multifunctional/theranostic PEGy-
lated polyester-based nanomedicine under investigation in

Pegylated Polyester-Based Na noncologicals Current Topics in Medicinal Chemistry, 2014, Vol. 14, No. 9 13
preclinical phase is represented by an engineered NP devel-
oped by Wang and colleagues [135]. NPs based on am-
phiphilic mPEG–PLGA and -polylysine (EPL) block co-
polymers were designed to co-deliver simoultaneously three
anticancer agents, doxorubicin packaged in the hydrophilic
core, paclitaxel inserted into the hydrophobic layer and sur-
vivin siRNA adsorbed on external surface. Finally, far red
fluorescent probes, such as fluorescein-isothiocyanate and
bovine serum albumin–Au were loaded into NPs, in order to
study in v ivo two-co lor tumor imaging. This promising sys-
tem demonstrated to accumulate predominantly into the tu-
mor regions of B16-F10 melanoma-bearing mice, where it is
able to exert an extremely effective antitumor efficacy, due
to the synergistic effects of the three drugs which target dif-
ferent essential metabolic pathways of tumor cells [135].
Recently, a series of biocompatible PEG-PLGA NPs for
multicolor and multiplexed imaging brighter than quantum
dots have been reported. More than 30 particle formulations
less than 100 nm in size and loaded with combinations of car-
bocyanine-based fluorophores (DiO, Dil, DiD, and DiR) that
exhibited distinct emission signatures (ranging from the visi-
ble to NIR wavelength region) were designed and tested [136].
A particle formulation that simultaneously emitted fluores-
cence at three different wavelengths upon a single excitation at
485 nm via sequential and multiple FRET cascade events for
multicolor imaging was identified. These NPs were individu-
ally conjugated with specific (Herceptin or IgG2A11 anti-
body) or nonspecific (heptaarginine) ligands for targeting and,
thus, could be applied to differentiate different cancer cells
from a cell mixture according to the expressions of cell-
surface human epidermal growth factor receptor 2 and the
receptor for advanced glycation endproducts. Using an animal
model subcutaneously implanted with the particles, it was
further demonstrated that the developed platform could be
useful for in vivo multiplexed imaging.
8. OTHER ADMINISTRATION ROUTES: A ROLE
FOR PEGYLATED NANONCOLOGICALS?
Despite the intravenous route remains the most direct and
efficient one to administer anticancer drugs, its intrinsic limi-
tations have prompted research interest in novel formulations
allowing the optimization of less invasive administration
routes. This is the case of oral and pulmonary delivery
routes, which have been recently exploited also for the ad-
ministration of PEGylated NCs.
The therapeutic efficacy of the majority of oral antican-
cer drugs appears seriously compromised by their poor oral
bioavailability, which can be increased through especially
engineered NCs [137, 138]. The intrinsic low permeability of
chemotherapeutics across gastrointestinal (GI) mucosa is due
to not only to the limited solubility (i.e. most anticancer
drugs show hydrophobic structure) and structural instability
in GI fluids, but also to the affinity for intestinal and liver
cytochrome P450 metabolic enzymes (first-pass extraction
mechanism) as well as to P-gp, which is involved in the
MDR [139, 140]. In this context, several works reported on
the ability of PEGylated nanoconstructs to incr ease the
bioavailability of oral anticancer drugs through by-passing
the active efflux of drugs mediated by intestinal P-gp [141-
145]. In fact, PEG has shown an inhibitory effect on the Pgp-
mediated efflux mechanism comparable to that obtained after
administration of verapamil, a calcium blocker and well-
known inhibitor of the P-gp efflux pump, usually associated
to chemotherapeutics in clinical oral therapy [146]. Follow-
ing this rationale, an amphiphilic conjugate of PEG 1 kDa
with a small molecule, D--tocopheryl succinate (Vitamin E
TPGS), has been explored to modify the surface of polyes-
ter-based NPs with encouraging in vitro/in vivo results [141].
One possible explanation of the Vitamin E TPGS superiority
can be its molecular weight, which seems optimal for the
inhibitory activity of PEG [147]. In fact, PEG molecular
weight can strongly influence the efficacy of oral nanomedi-
cines and, usually, the lower is PEG molecular weight the
higher is oral bioavailability [141].
Concerning oral delivery, PEGylated NPs may also rep-
resent an effective strategy to protect the drug toward liver
cytochrome P450 metabolic enzymes [141] and GI harsh
enzymes [148]. In particular, PEG-PLA NPs conferred
higher stability to encapsulated antigen in simulated GI flu-
ids containing digestive enzymes (i.e. pepsin and pancreatin)
as compared with non-modified PLA NPs [148]. The
mechanism of PEG stabilization effect is not clear, but it can
be reasonably attributed to a reduced interaction between the
NP and the enzymes of the digestive fluids due to PEG
cloud. By the same mechanism, an increased systemic ab-
sorption of curcumin administered orally through PEGylated
PLGA NPs was achieved in vivo [149]. Another important
implication of using PEGylated NPs for oral delivery is re-
lated to their potential to cross the loosely adheren t mucus
layer, which is continuously removed by peristalsis and re-
placed, and to reach the firmly adherent layer deposed on GI
epithelium [150]. Hanes and co-workers have recently dem-
onstrated that mucoinert PEGylated NCs of adequate size
(200-500 nm), the so-called Mucus-Penetrating Particles
(MPP), may deeply penetrate human mucus, whereas com-
parably sized uncoated particles are immobilized by the mu-
cus meshes [151].
The potential of MPP to penetrate mucus barrier makes
PEGylated polyesters NPs interesting also for pulmonary
delivery, where the role of extracellular barriers on inhaled
drug bioavailability is nowadays well acknowledged [152].
The well-establised ability of PEGylated NPs to escape
macrophage uptake can be also useful to increase persis-
tency of the drug carrier at lung. Finally, the contemporary
local and sustained release of the entrapped drug cargo may
allow to reduce the number of administrations and, in so
doing, side effects. Although pulmonary delivery of drug-
loaded NPs for lung cancer treatment is still in its infancy
[153-155], some proof of concepts of the safety/efficacy of
PEGylated polyester-based particles for pulmonary deliv-
ery can be found in recent literature. The feasibility of
PEG-PLGA copolymers as carriers for pulmonary delivery
of a highly soluble drug (low molecular weight heparin)
was assessed in vitro and in vi vo [156]. No cytotoxic effect
was observed when bronchial epithelial cells were incu-
bated with PEG-PLGA based formulations. Similarly, no
increase in th e injury markers was observed in the bron-
choalveolar lavage fluids collected from rats treated with
PEG-PLGA particles. Overall, this study suggests that
PEG-PLGA block copolymers have the potential for pul-
monary delivery of drugs. More recently, PEG-PDLLA

14 Current Topics in Medicinal Chemis try, 2014, Vo l. 14, No. 9 Conte et al.
NPs have been preliminarly investigated for paclitaxel
palmitate delivery in lung cancer treatment [157]. From a
therapeutic point of view, it has been observed that the con-
jugation of PEG-PLA NPs to a targeting ligand, the epithe-
lial cell adhesion molecule (EpCAM, CD326), may further
promote specificity of the carrier cargo to cancer cells, lim-
iting potential drug-related toxicity effects on healthy cells.
Pulmonary delivery of drug loaded nano- and/or immune
NP formulations elicited mild inflammation as evidenced
by the slightly increased neutro phil and activated macro-
phage counts in bronchoalveolar lavage. No evidence for
pulmonary toxicity following treatment with either blank or
drug-loaded nano- and/or immunonanoparticles was ob-
served.
An open issue remains the way to deliver NPs at lung,
which is generally accomplished by poorly stable aqueous
dispersions to be delivered through time-consuming and of-
ten bulky nebulizers [158, 159]. A current paradigm is that
NPs may be delivered directly to the lung in form of dry
powders by means of handy breath-actuated dry powders
inhalers (DPIs) [155]. Since the greatest challenge is the low
inertia of particles with a mean size lower than 1 μm (ex-
haled upon inhalation), the engineering of NPs at the mi-
crosize level, thus obtaining “micrometric” NP-based dry
powders for inhalation has been envisaged [160, 161]. In so
doing, all the advantages of PEGylated NPs (e.g., macro-
phage escape, long-term residence in situ; prolonged drug
release;) could be combined with the ease of use of DPIs. In
this context, large porous carriers (so called “Trojan parti-
cles”) [162] or microparticles based on inert materials (so-
called “nano-embedded microparticles”) [163, 164] have
been recently developed.
This less-inv asive route of administration might change
the way lung cancer is treated in the future. Nevertheless,
one should be aware of potential health and safety risks
associated with handling cytotoxic dry powders in the
healthcare industry. Of course, special attention must be
paid to the selectivity of the system for lung cancer cells as
well as the long-term safety of the polymer carrier in th e
lung.
CONCLUSIONS
Functionalized PEGylated polyesters offer a versatile
tool to impart a wide range of properties to nanosized carri-
ers. Rationale combination of different building elements
can produce “smart” systems moulded on peculiar tumor
features and able to overcome complex biological barriers
in the body. In particular, the selection of the base material
(chemistry, molecular weight, architecture) combined with
adequate preparation methods and careful characterization
in biologically relevant conditions will be useful to drive
NC design with the final aim to improve the outcome of
cancer therapy. Only if a rational carrier development is
carried out, the translation of PEGylated polyester-based
nanomedicines in the treatment of cancer malignancies will
be successful.
CONFLICT OF INTEREST
The authors confirm that this article content has no con-
flicts of interest.
ACKNOWLEDGEMENTS
The authors thank the Italian Ministry of University and
Research (MIUR, PRIN project 2010-2011) for funding sup-
port.
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Received: December 18, 2013 Revised: January 28, 2014 Accepted: February 26, 2014