Doxorubicin

orubicin
Review
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pH-sensitive polymeric nanoparticles for

tumor-targeting doxorubicin delivery:
concept and recent advances
Doxorubicin is a potent chemotherapeutic drug applied in the clinics for the treatment
of various human cancers. It is typically administered as the hydrochloride salt or in
liposomal forms, which are plagued with severe side effects. In recent years, pHsensitive polymeric nanoparticles that are capable of retaining drug during circulation
while actively releasing it at the tumor site and/or inside the target tumor cells have
received an overwhelming interest for tumor-targeting cancer chemotherapy. This
smart delivery approach has shown to elegantly resolve the in vivo stability versus
intracellular drug release dilemma, as well as stealth versus tumor cell uptake dilemma.
In this review, the concept and exciting new advances in pH-sensitive polymeric
nanoparticles for doxorubicin delivery are presented and discussed.
Keywords: cancer chemotherapy cancer stem cells controlled release doxorubicin
multidrug resistance pH-sensitive polymeric nanoparticles
Background
Doxorubicin (DOX), also known as adriamycin (ADR), is a potent chemotherapeutic
drug applied in the clinics for the treatment
of a wide range of human cancers, including Hodgkins lymphoma, leukemia, multiple myeloma, breast cancer, osteosarcoma,
ovarian cancer and lung cancer. Like other
anthracyclines, DOX takes effect by intercalating DNA in cancer cells and inhibiting macromolecular biosynthesis. DOX is
typically administered intravenously as the
hydrochloride salt (DOX HCl) or in liposomal forms. The hydrochloride salt formulations (available under the brand names Adriamycin PFS, Adriamycin RDF and Rubex)
that show a high activity against various solid
tumors with a good therapeutic index are
plagued with severe side effects, such as heart
damage, typhlitis, heart arrhythmias, nausea
and vomiting. Liposomal DOX (available
as Doxil, Caelyx and Myocet) has shown
clearly reduced cardiotoxicity as compared
with the unencapsulated DOX. It is found,
however, that PEGylated liposomal formulations might cause handfoot syndrome that
10.2217/NNM.13.212 2014 Future Medicine Ltd
Fenghua Meng1,
YinanZhong1,
RuCheng1, Chao Deng1
&ZhiyuanZhong*,1
1
Biomedical Polymers Laboratory
&Jiangsu Key Laboratory of Advanced
Functional Polymer Design & Application,
College of Chemistry, Chemical
Engineering & Materials Science,
Soochow University, Suzhou, 215123,
Peoples Republic of China
*Author for correspondence:
Tel.: +86 512 65880098
Fax: +86 512 65880098
zyzhong@ suda.edu.cn
limits its dose and substitution for DOX HCl

formulations.
In the past decade, stealth polymeric
nanoparticles such as micelles and polymersomes have emerged as a most promising technological platform for DOX delivery [13] . Unlike liposomes that have to be
modified with poly(ethylene glycol) (PEG)
to obtain stealth liposomes, polymeric
nanoparticles are intrinsically shielded
by nonfouling polymers. Moreover, they
also offer benefits of better stability, low
immunogenicity and versatile structures
and functions as compared with liposomes.
With proper particle size and surface
chemistry, polymeric nanoparticles have
been shown to be able to circulate invivo
for a prolonged time, effectively alleviate
adverse effects including cardiotoxicity,
improve drug bioavailability and enhance
drug tolerance. Notably, a few polymeric
DOX prodrugs (PK1 [4] and PK2 [5] ) and
micellar DOX formulations (NK911 [6] ,
and SP1049C [7]) have advanced to different phases of clinical trials. However,
despite significant progress, as well as great
Nanomedicine (2014) 9(3), 487499
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ISSN 1743-5889
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Review Meng, Zhong, Cheng, Deng & Zhong

promise, nanoparticulate DOX formulations have
met with limited success due to existing extra- and
intra-cellular barriers, such as poor in vivo stability, short circulation time, modest accumulation in
the tumor, inefficient uptake by tumor cells or slow
intracellular drug release [810] .
As an ideal drug vehicle, polymeric nanoparticles
should be able to retain cargo during circulation
while dumping all the drugs upon arriving at the
site of action. Because DOX takes therapeutic effect
by intercalating DNA, drug should be delivered and
released into the nuclei of target cancer cells. In the
recent several years, much research effort has been
directed to the development of pH-sensitive polymeric nanoparticles for intracellular DOX delivery in that there exist natural pH gradients in the
tumor microenvironment (pH 6.57.2) [11] and in
the endosomal/lysosomal compartments of tumor
cells (pH 4.06.5) [12] . It is interesting to note that
a pH responsiveness strategy has been exploited
to overcome various extra- and intra-cellular barriers to successful cancer chemotherapy for nanoscale
DOX delivery systems (Figure 1) . For example, taking advantage of acidic extracellular pH (6.57.2) in
the tumor compared with the normal tissues, super
pH-sensitive nanoparticles have been developed to
achieve accelerated drug release at the tumor site.
pH-responsive nanoparticles that reverse charge or
expose ligands at the outer surface (deshielding) at
tumor pH have been designed to facilitate tumor cell
uptake. Acid-sensitive nanoparticles that are prone to
swelling, dissolution or degradation at endosomal/
lysosomal pH (4.06.5) have been devised to obtain
fast intracellular DOX release in tumor cells. Moreover, pH-sensitive cross-linked nanoparticles have
been proposed to resolve the extracellular stability
and intracellular DOX release dilemma. These pHsensitive nanoparticulate DOX formulations have
demonstrated markedly improved antitumor activity
in vitro and/or in vivo as compared with their pHinsensitive counterparts [13,14] . It should further be
noted that a fast DOX release feature renders pHsensitive nanoparticles also particularly appealing for
treatment of multidrug-resistant (MDR) cancers. In
addition to its high potency, DOX possesses unique
solubility and fluorescence properties, which make it
an ideal model for both hydrophobic (DOX in free
base form) and hydrophilic (DOX in hydrochloride
salt form) anticancer drugs. In this review, exciting
new advances in pH-sensitive polymeric nanoparticles for DOX delivery are presented and discussed.
At the end, a conclusion and future perspective on
pH-sensitive nanoparticulate DOX formulations for
cancer chemotherapy are given.
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Nanomedicine (2014) 9(3)
Endosomal/lysosomal pH-sensitive
polymeric nanoparticles for active
intracellular DOX delivery
Polymeric nanoparticles are usually internalized by
cancer cells via endocytosis. Following endocytosis,
rapid endosomal acidification occurs due to a vacuolar
proton ATPase-mediated proton influx, which leads to
a drop of pH levels in the endosomes to approximately
5.06.5 and 4.05.0 in the lysosomes [12] . In the past
few years, this acidic pH in the endosomal/lysosomal
compartments has been utilized as an effective means
to trigger intracellular DOX release from acid-sensitive
nanoparticles in order to enhance the therapeutic efficacy and reverse multidrug resistance in tumors [15] .
It is worthy to note that pH-sensitive nanoparticles
may also facilitate disruption of endosomes via a proton sponge effect or interaction with the endosomal
membrane.
pH-sensitive nanoparticles that are prone to swelling or dissolution at endosomal/lysosomal pH have
been designed based on polymers containing protonable amine groups, such as primary, secondary
and tertiary amines. For instance, Kim et al. reported
that DOX-loaded micelles based on poly(2-hydroxyethyl methacrylate)-b-poly(l-histidine) (p(HEMA)b-p(His)) released DOX in a pH-dependent manner
and induced higher growth inhibition of human colon
tumor 116 human colon carcinoma cells at acidic than
basic pH [16] . He et al. reported that DOX-loaded
PEG-poly(l-histidine)-poly(l-lactic acid) (PEG45PHis45-PLLA82) nanoparticles would swell and promote DOX release at pH 5.0 due to protonation of the
imidazole groups in the PHis block, inducing a high
antitumor effect in HepG2 cells [17] . Qiu et al. demonstrated that DOX-loaded pH-sensitive micelles based
on polyphosphazene-containing diisopropylamino
(DPA) side groups had similar antitumor activity to
free DOX against MCF-7 cells and a 1020-fold lower
IC50 than free DOX against drug-resistant MCF-7 cells
(MCF-7/ADR) [18] . The high antitumor efficacy was
due to pH-triggered DOX release, as well as enhanced
endosomal escape by the proton sponge effect of DPA
moieties. Zhou et al. reported that N-deoxycholic acidO, N-hydroxyethylation chitosan micelles modified
with octreotide-PEG-deoxycholic acid exhibited pHdependent DOX release, efficient uptake by MCF-7
cells (overexpressing somatostatin receptors) via receptor-mediated endocytosis and enhanced antitumor
efficacy as compared with nontargeting N-deoxycholic
acid-O, N-hydroxyethylation chitosan micelles in nude
mice bearing MCF-7 cancer xenografts [19] .
pH-sensitive nanoparticles have also been developed
by incorporating acid-cleavable bonds such as hydrazone, acetal, imine and oxime bonds onto polymer main
future science group
pH-sensitive polymeric nanoparticles for tumor-targeting doxorubicin delivery: concept & recent advances
Tumor tissue
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Review
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Targeting ligand (TAT, biotin)
Acid-cleavable bonds: hydrazone,
Protecting moiety
acetal, cis-acotinyl, imine and oxime
Nanomedicine Future Medicine Ltd (2014)
Figure 1. Design and development of pH-sensitive polymeric nanoparticles for doxorubicin delivery. (A) Tumor
extracellular pH-sensitive nanoparticles to facilitate tumor cell uptake and/or drug release at the tumor site.
(i) Tumor pH-triggered surface charge conversion (i.e., from negative or neutral to positive charge); (ii) tumor pHtriggered pop-up of specific ligands; (iii) tumor pH-triggered deshielding of CPP from the nanoparticle surface;
and (iv) tumor pH-triggered nanoparticle dissociation and DOX release. (B) Endosomal/lysosomal pH-sensitive
nanoparticles to accomplish fast intracellular drug release and disruption of endosomes. (v) Endosomal/lysosomal
pH-triggered swelling and dissolution of nanoparticles; and (vi) endosomal/lysosomal pH-triggered degradation
of nanoparticles.
DOX: Doxorubicin; TAT: Trans-activator of transcription.
or side chains. For example, van Hest et al. reported

that polymersomes based on PEG-b-polybutadiene
connected by a hydrazone bond displayed a strong pHdependent colloidal stability due to pH-induced shedding of PEG mantles [20] . Yang et al. found that the
intervening benzoic-imine linker in PEG-C18 block
copolymer micelles exhibited pH-dependent progres-
sive hydrolysis behavior [21] . The imine linker, while

stable at physiological pH, was partially hydrolyzed to
present positive surface charge at tumor pH, facilitating tumor cell uptake, and completely hydrolyzed at
endosomal pH, resulting in micelle dissociation, fast
intracellular DOX release and effective membrane
disruption. Zhu et al. reported that DOX release from
www.futuremedicine.com
489

biodegradable micelles self-assembled from oximetethered PEG-PCL (poly(-caprolactone)) block
copolymers was significantly accelerated at pH 5.0 as
compared with physiological pH, resulting in a high
anticancer efficacy in HeLa cells [22] . Along with reduction-sensitive shell-sheddable micelles and polymersomes [2325] , triggered shell-shedding has appeared as a
most straightforward and effective approach to achieving efficient intracellular drug release. Recently, we
discovered that core-cross-linked polypeptide micelles
based on lipoic acid and cis-1,2-cyclohexanedicarboxylic acid (CCA)-conjugated PEG-poly(l-lysine) block
copolymer had enhanced DOX loading at neutral pH
due to the presence of ionic and hydrogen bonding
interactions with CCA, but an accelerated drug release
at endosomal pH owing to cleavage of the acidic-labile
amide bond between CCA and PLL [26] . Interestingly,
these dual-bioresponsive micelles showed more efficient nucleus delivery of DOX and thereby better antitumor activity than their pH-insensitive counterparts.
Frchet et al. disclosed that micelles formed from PEO
(poly(ethylene oxide))-dendritic polyester copolymer
with acid-labile acetal groups on the core-forming dendrimer periphery exhibited high pH sensitivity [27] . The
in vitro release studies showed that DOX release was
highly pH dependent. Inspired by the work of Frchet,
we have prepared pH-sensitive degradable micelles and
polymersomes based on acetal-containing polycarbonate, poly(2,4,6-trimethoxybenzylidenepentaerythritol
carbonate), as a hydrophobe [28,29] . The in vitro drug
release studies showed that DOX and DOX HCl were
released in a pH-dependent manner from micelles
and polymersomes, respectively. In a following study,
pH-sensitive degradable chimeric polymersomes were
developed from PEG-poly(2,4,6-trimethoxybenzylidene-1,1,1-tris (hydroxymethyl) ethanemethacrylate)poly(acrylic acid) for efficient loading of DOX HCl
into the interior of polymersomes at neutral pH via
the electrostatic/hydrogen bonding interactions, as
well as triggered drug release at pH 5.4 through acetal
hydrolysis (Figure 2) [30] . DOX HCl-loaded chimeric polymersomes revealed a high antitumor activity
comparable with free DOX HCl in HeLa cells.
The development of endosomal pH-activatable macromolecular prodrugs and prodrug nanoparticles with
DOX covalently conjugated to the polymer chains via
a cleavable hydrazone or cis-aconityl bond represents
another interesting approach to pH-sensitive DOX
delivery [31] . As from the 1980s, polymeric prodrugs
have received tremendous interest for cancer chemotherapy. Unlike self-assembled nanovehicles that tend to
dissociate and release encapsulated drug upon intravenous administration, polymeric prodrugs are generally
stable in blood circulation and may effectively prevent
490
premature drug release as a result of covalent linking

of the drug to the carrier. We found that pH-sensitive
PEO-g-DOX prodrugs with DOX linking to the PEO
main chain via a hydrazone bond while sufficiently stable at pH 7.4 were readily activatable at endosomal pH
[32] . Haag et al. reported that DOX prodrugs based on
dendritic polyglycerol via a cleavable hydrazone bond
and with PEG shells exhibited high drug loading, good
water solubility, pH-dependent drug release behavior
and improved antitumor efficacy over free DOX in an
ovarian xenograft tumor model (A2780) [33] . In a recent
study, immunoprodrugs were developed from a polyglycerolDOXPEG construct by conjugating a scFv
antibody to the terminal of the PEG shell using SNAPTag (human DNA-repair enzyme O-6-alkylguanine
DNA alkyltransferase) technology [34] . These immunoprodrugs exhibited pronounced targetability and
specific toxicity toward EGF receptor-overexpressing
cancer cells, such as A431, MDAMB-468 and Panc-1
cells. The current polymeric prodrugs are usually based
on relatively low molecular weight (<45 kDa) water-soluble polymers because higher molecular weight polymers can not readily be excreted from the body. The
low molecular weight macromolecular drugs, however,
exhibit relatively short circulation time and poor accumulation in the tumor in vivo.
In recent years, prodrug micelles with DOX covalently conjugated to the micellar core, which elegantly
combine the stability of prodrugs with the long circulation time of micelles, were actively developed for
improved cancer therapy. Park et al. prepared acidactivatable micellar DOX prodrugs through conjugating DOX to a PEG-PLA (polylactide) terminal
via a pH-sensitive hydrazone or cis-aconityl bond [35] .
Kataoka et al. obtained pH-sensitive micellar prodrugs
from PEG-polyaspartate block copolymer grafted with
DOX via a hydrazone linkage [36,37] . Ulbrich et al.
found that DOX prodrug micelles with DOX covalently conjugated to the PEO-b-poly(allyl glycidyl
ether) micelle core via a hydrazone bond had reduced
systemic toxicity and pronounced therapeutic activity
in murine EL-4 T-cell lymphoma-bearing mice [38,39] .
Jing et al. reported that folate-decorated DOX prodrug micelles with hydrazone linkage showed better
pH sensitivity as compared with those with carbamate
linkage and pronounced targeting and cytotoxicity to a
human ovarian cancer cell line (SKOV-3) [40] . Further
in vivo studies in H22 tumor-bearing mice showed
that tumor-targeting DOX prodrug micelles resulted
in efficacious tumor growth inhibition [41] . Gong
et al. reported that superparamagnetic iron oxide/
DOX-loaded vesicles based on asymmetric FA-PEG114P(Glu-Hyd-DOX)-PEG46 -acrylate triblock copolymer
could be cross-linked to achieve enhanced stability,
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DOX-HCl-loaded acid-labile
chimeric polymersome
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of polymersome and drug release
Figure 2. pH-sensitive degradable chimeric polymersomes based on asymmetric poly(ethylene glycol)-poly(2,4,6trimethoxybenzylidene-1,1,1-tris (hydroxymethyl) ethanemethacrylate)-poly(acrylic acid) triblock copolymers (poly[ethylene glycol]
block longer than poly[acrylic acid] block) for active loading, as well as endosomal pH-triggered release of doxorubicinhydrochloride
salt.
DOX HCl: Doxorubicinhydrochloride salt; PAA: Poly(acrylic acid); PEG: Poly(ethylene glycol); PTTMA: Poly(2,4,6trimethoxybenzylidene-1,1,1-tris [(hydroxymethyl] ethanemethacrylate).
Reproduced with permission from [30] .
exhibited a much higher r2 relaxivity value than Feridex (a commercial superparamagnetic iron oxide-based
T2 contrast agent), released DOX under mildly acidic
pH and caused higher cytotoxicity than folate (FA)free vesicles [42] . Gu et al. reported that pH-sensitive
nanoparticles formed from PEGylated peptide dendronDOX conjugates with 14.0 wt.% drug loading
displayed much faster DOX release at pH 5.0 than 7.4
due to acid cleavage of the hydrazone bonds [43] . The
in vivo studies showed that these DOX nanoparticles
caused no significant systemic toxicity, but had strong
antitumor activity in the 4T1 breast tumor model. It
should be noted, nevertheless, that DOX release from
these prodrug nanoparticles is often slow and incomplete (<50%), even after several days, probably due
to retarded hydrolysis and drug diffusion from the
hydrophobic micellar core. To this end, we designed
pH-activatable DOX prodrug nanogels by conjugating

DOX to PEG-b-poly(2-hydroxyethyl methacrylate-coethyl glycinate methacrylamide) copolymers via hydrazone bonds [44] . These prodrug nanogels released DOX
nearly quantitatively over 48 h at endosomal pH due
to excellent permeability and caused pronounced cytotoxic effects on RAW 264.7 and MCF-7 tumor cells.
Notably, Kratz et al. developed an acid-sensitive DOX
prodrug, the (6-maleimidocaproyl)hydrazone derivative of DOX (INNO-206, formerly DOXO-EMCH),
which could rapidly bind to circulating serum albumin
while releasing DOX selectively at the tumor site [45,46] .
This albumin-binding DOX prodrug showed superior
antitumor activity to DOX in different xenograft models (such as breast carcinoma 3366, ovarian carcinoma
A2780 and small-cell-lung cancer H209), as well as in
an orthotopic pancreas carcinoma model (AsPC-1) [46] .
491

It is interesting to note that INNO-206 has already
been advanced to Phase II clinical studies [45] .
The rapid intracellular drug release also renders pHsensitive DOX nanoparticles effective against MDR
cancer cells and in vivo. For example, Nagasaki et al.
reported that DOX-loaded PEGylated nanogels containing a pH-sensitive polyamine core exhibited superior antitumor activity against drug-resistant human
hepatoma HuH-7 cells to their free DOX and pHinsensitive counterparts [47] . The pH-sensitive DOXloaded PEG-poly(4-vinylbenzylphosphonate) nanoparticles displayed much higher cytotoxic activity against
both P-gp- and MRP1-overexpressing cells than free
DOX [48] . Wang et al. found that gold nanoparticles
with surface-tethered DOX via a PEG spacer through
a pH-sensitive hydrazone linkage achieved enhanced
drug accumulation and retention in multidrug-resistant MCF-7/ADR cancer cells, resulting in effective
reversal of multidrug resistance [49] . Qiu et al. demonstrated that DOX-loaded pH-sensitive micelles based
on polyphosphazene-containing DPA side groups
caused a 1020-fold lower IC50 than free DOX against
drug-resistant MCF-7/ADR cells [18] . Lavasanifar and
Xiong reported that tumor-targeting micellar nanoparticles based on PEO-PCL block copolymers with PCL
end-functionalized using either a short polyamine for
siRNA complexation or a DOX molecule through a
pH-sensitive hydrazone linkage were able to simultaneously deliver DOX and siRNA against P-gp expression
into multidrug-resistant MDA-MB-435 human tumor
models, leading to effective reversal of drug resistance
[50] . Bae et al. disclosed that DOX-loaded micelles
based on PEG-P(His-co-phenylalanine) and PEGPLA-folate block copolymers, which targeted to folate
receptors and early endosomal pH, effectively suppressed the growth of MDR ovarian tumor xenograft
in mice for at least 50 days without weight loss [14] .
Tumor pH-sensitive polymeric nanoparticles
for DOX delivery
The cancerous tissue is slightly acidic with pH values
ranging from 6.5 to 7.2 due to a combination of elevated aerobic glycolysis and reduced blood flow. In the
past few years, super pH-sensitive nanoparticles that
are sufficiently stable at a physiological pH of 7.4 but
are prone to deshielding, which thereby facilitate tumor
cell uptake or swelling, thereby triggering drug release,
have been developed for tumor-targeting drug release
[5153] . Tumor extracellular pH-sensitive nanoparticles
are typically designed based on polymers with pK a in
the range of 6.5 to 7.2, such as poly(l-histidine) (PHis)
and poly(-amino esters) (PAE). Bae et al. prepared
ultra pH-sensitive micelles based on PEG-PHis and
PEG-PLA block copolymers that were fairly stable at
492
pH 7.07.4, whereas they were disassembled at a pH of

6.67.2. Notably, folate-decorated DOX-loaded ultra
pH-sensitive micelles showed a high cytotoxicity to
drug-resistant MCF-7 cells in vitro and in vivo [54] . Lee
et al. discovered that pH-responsive PEG-PAE micelles
exhibited a sharp micellization/demicellization transition at pH 6.46.8 [55] . DOX-loaded PEG-PAE micelles
afforded enhanced tumor growth inhibition and prolonged survival of B16F10 tumor-bearing mice as compared with free DOX. In a subsequent study, tumortargeting pH-responsive micelles were constructed
from PEG-PAE and AP (CRKRLDRN)-PEG-PLA
[56] . The in vivo studies in mice bearing human breast
MDA-MB231 tumor (overexpressing AP-specific IL-4
receptors) showed that DOX-loaded targeting micelles
had better tumor accumulation and in vivo therapeutic efficacy than their nontargeting counterparts and
free DOX. Poly(N-(3-diethylamino)propyl isothiocyanato-l-lysine)-PEG-PLLA triblock copolymer formed
flower-like micelles that were stable at pH 7.4 but disintegrated at pH <7.0 [57] . The in vitro release studies
showed that DOX release from flower-like micelles was
much accelerated by tumor pH.
Tumor extracellular pH-sensitive nanoparticles have
also been constructed by reversibly shielding the positive
surface charges (usually primary amines) or ligands (e.g.,
trans-activator of transcription [TAT]) using tumor
pH-cleavable bonds such as cis-aconityl, imine, oxime
and succinyl amide. This tumor pH-sensitive reversible
shielding represents an elegant approach to addressing
the dilemma of stealth versus tumor cell uptake. Wang
et al. reported that dual pH-sensitive nanoparticles of a
polymerDOX conjugate (PPC-Hyd-DOX-DA) could
reverse the surface charge from negative to positive at
pH 6.8 by acid-responsive cleavage of -carboxyl amide
bonds, facilitating tumor cell internalization and DOX
release at endosomal pH owing to hydrazone cleavage
[58] . These dual pH-responsive DOX prodrug nanoparticles demonstrated enhanced inhibition of drug-resistant SK-3rd cancer stem cells. The same group showed
that a DOX-loaded charge-conversional nanogel system
displayed an approximately 30-fold higher internalization and almost two-fold higher antitumor activity
against MDA-MB-435s cells at pH 6.8 than 7.4 [59] .
Du et al. reported that chitosanstearic acid conjugate nanoparticles PEGylated with an acid-sensitive
cis-aconityl linkage showed accelerated DOX release
and enhanced cellular uptake under weakly acidic
environments [60] . The in vivo studies in BEL-7402
tumor-bearing mice revealed much more accumulation
of pH-sensitive PEGylated nanoparticles in the tumor
and better antitumor performance than acid-insensitive
PEGylated controls. This tumor pH-triggered deshielding offers an efficient and general strategy for solid
tumor targeting, which is an appealing alternative to

cell-specific active targeting.
Tumor pH-triggered pop-up or deshielding of CPP
from the nanoparticle surface has been proposed to
facilitate translocation of nanoparticles into tumor
cells. Bae et al. reported that micelles self-assembled
from PLLA-PEG-PHis-TAT and PEG-PHis copolymers hid TAT at physiological pH, but exposed it at
a slightly acidic tumor extracellular pH [61] . Moreover, the micelle core was designed to disintegrate and
quickly release DOX at endosomal pH. These super
pH-sensitive nanoparticles augmented DOX potency
in various wild and multidrug-resistant cell lines with
a 3.88.8-fold lower IC50 than free DOX. The in vivo
studies in nude mice bearing either drug-sensitive or
-resistant human xenografts all showed significant
tumor regression with minimum weight loss. pH-sensitive micelles with reversibly shielded TAT ligands were
also developed from TAT-PEG-PLLA and pH-sensitive
PEG-poly(methacryloyl sulfadimethoxine) diblock
copolymer, in which anionic poly(methacryloyl sulfadimethoxine) complexed with cationic TAT of the
micelles at physiological pH to yield stealth micelles
[62] . However, at pH 6.6, TAT would be exposed on
the micelle surfaces, leading to significantly higher
cellular uptake as compared with pH 7.4, supporting
shielding of TAT-PEG-PLLA micelles at normal pH
and deshielding at tumor pH. Very recently, Shen et al.
modified the TAT lysine residues in TAT-PEG-PCL
micelles using pH-sensitive succinyl amides to inhibit
nonspecific interactions of TAT in the bloodstream
(Figure 3) [63] . In acidic tumor or endosomal/lysosomal
compartments, succinyl amides would be quickly
hydrolyzed to fully restore TATs functions. These pHsensitive targeting micelles achieved long circulation
in the blood and efficiently accumulated and delivered
DOX to tumor tissues, giving rise to high antitumor
activity and low cardiotoxicity.
In addition to TAT ligand, tumor pH-triggered
pop-up of specific ligands (e.g., biotin) has been investigated to achieve quick tumor cell uptake via receptormediated endocytosis. For instance, Bae et al. reported
that pH-sensitive micelles based on PEG-PHis and
biotin-PHis-PEG-PLA block copolymers were shielded
by PEG at pH 7.4 with biotins hiding at the coreshell
interface [64] . At tumor pH (pH 7.0), PHis became
water soluble, exposing biotin on the micelle surface
and facilitating cell uptake via receptor-mediated
endocytosis. Notably, these micelles also exhibited pHdependent dissociation and enhanced DOX release at
endosomal pH. Recently, Li et al. prepared pH-sensitive nano-flower micelles that exhibited a half-open
state to expose biotin for efficient cellular uptake at
tumor pH due to acidic cleavage of the benzoicimine
Review
bond and fully bloomed to release DOX at endosomal

pH owing to cleavage of hydrazone bonds [65] .
pH-sensitive cross-linked polymeric
nanoparticles to resolve the extracellular
stability and intracellular DOX release
dilemma
One practical challenge with nanoparticulate drug formulations is their low in vivo stability due to large volume
dilution and/or interactions with cells and biomolecules
present in the blood, which often leads to premature drug
release, aggregation and diminished drug targetability
[66,67] . The chemical cross-linking of nanoparticles, however, can improve their stability results in usually reduced
drug efficacy due to prohibited drug release at the target
sites. pH-sensitive cross-linked nanoparticles have been
proposed to resolve the extracellular stability and intracellular drug release dilemma [68] . Lee et al. reported that
ketal cross-linked micelles based on PEG-PAsp-poly(lphenylalanine) had improved stability against micelledisrupting sodium dodecyl sulfate surfactant [69] . The
release of DOX was rapid at endosomal pH as compared
with physiological pH due to reversal of cross-linking
via acidic degradation of ketal linkages (ketal hydrolysis
half-life: 0.7 h at pH 5.0 vs 52 h at pH 7.4). The confocal observations showed that ketal-cross-linked micelles
could efficiently release DOX in endosomes as well as
into the nuclei of MCF-7 cells over 5 h. In a following
study, PEG-PAsp-poly(l-phenylalanine) micelles were
stabilized by calcium phosphate (CaP) that grew in the
anionic PAsp shell domains (Figure 4) [70] . CaP-stabilized
micelles displayed good serum stability. The release of
DOX from CaP-stabilized micelles was inhibited at pH
7.4 but significantly enhanced at pH 4.5 due to rapid
dissolution of CaP mineral layers. The in vivo studies in
MDA-MB231 tumor-bearing mice showed that DOXloaded CaP-stabilized micelles exhibited prolonged
circulation, enhanced tumor specificity and better
therapeutic efficacy compared with free DOX and nonstabilized controls. Zeng et al. discovered that pH-sensitive micelles assembled from P[PEGMA-b-(DEMAco-APMA)]-FA that contains adenine (A) and tertiary
amine moieties in the hydrophobic block, and FA targeting ligand at the terminal of hydrophilic block, could be
cross-linked using uracil-(CH2)6-uracil via A-U nucleobase pairing based on complementary multiple hydrogen
bonding at neutral pH [71] . These cross-linked micelles,
while stable at physiological pH, were dissociated under
acidic pH due to protonation of tertiary amines (pKa
6.47.0) and disruption of the adenineuracil nucleobase pairing. DOX-loaded micelles were preferentially
taken up by folate receptor-positive cancer cells. Shuai et
al. prepared pH-sensitive interlayer-cross-linked micelles
from mPEG-PAsp(MEA)-PAsp(DIP)) triblock copoly-
493
Blood
Tumor tissue
Tumor cell
NPC
Pore
Acidic extracellular fluid
Lysosome
(pH 45)
O
Cys
A rg
A rg
A rg
NH
= aTAT
O
O
Amidization
(deactivation)
CH
A rg
A rg
O
CH
H
N
CH2
CH2
CH2
CH2
CH2
CH2
NH
CH2
CH2
NH
NH
C
CH2
CH2
CH2
CH2
O
A rg
A rg
NH
= TAT
O
CH
A rg
A rg
H
N
G ly T yr
C
OH
O
CH
CH2
OH
A rg
CH
CH2
OH
Cys
CH2
CH2
Acidic pH
(activation)
H
N
H
N
O
CH
CH2
CH2
CH2
CH2
CH2
CH2
CH2
CH2
NH2
CH2
CH2
NH2
NH2
G ly T yr
Figure 3. Acid-active cell-penetrating peptides for in vivo tumor-targeted drug delivery. (A) Deactivation of TAT in the blood
compartment and its activation in the tumor interstitium or cells for in vivo tumor-targeted drug delivery. The amines of the lysine
residues of TAT are amidized to inhibit its nonspecific interactions in the blood compartment without affecting the nanocarriers
stealth properties. Once the nanocarrier extravasates into tumor tissue through highly permeable blood vessels via the enhanced
permeability and retention effect, these amides are hydrolyzed, regenerating the pristine functioning CPP in the acidic tumor
extracellular fluids (pH <7) for fast cellular uptake or in acidic endosomes/lysosomes, for fast endosomal/lysosomal escape and nuclear
targeting. (B) Amidization of TATs primary amines to succinyl amides and their acid-triggered hydrolysis.
NPC: Nuclear pore complex; TAT: Trans-activator of transcription.
494
mers, which were stable and free of drug leakage at neutral pH [72] . The release of DOX was, however, accelerated at pH 5.0. The in vivo studies in nude mice bearing
the Bel-7402 xenograft demonstrated reduced premature
drug release in blood circulation and better therapeutic
effects than DOX-loaded PEG-PCL micelles and free
DOX. Hennink et al. reported that core-cross-linked
DOX prodrug micelles based on mPEG-b-p(HPMAmLacn) released drug completely within 24 h at pH 5 and
37C [73] . In comparison, only approximately 5% DOX
was released at pH 7.4 under otherwise the same conditions. These cross-linked DOX prodrug micelles demonstrated better antitumor activity than free DOX in mice
bearing B16F10 melanoma carcinoma.
PEG
PAsp PPhe
CH3 OCH2CH2 113NH COCHNH
12
COCHNH
Tumor extracellular pH-sensitive nanoparticles

have been designed to achieve accelerated drug
release at the tumor site;
pH-responsive nanoparticles that reverse surface
charge or expose ligands such as TAT and biotin at
Ca2+
DOX
O
Inorganic CaP layer

PO43-
23
CH2
CH2
C
Conclusion & future perspective

The past few years have witnessed remarkable advances
in pH-sensitive nanoparticles for tumor-targeting
DOX delivery. The pH-responsiveness has been
employed as an elegant and unique strategy to overcome various extracellular and intracellular barriers to
successful cancer chemotherapy for nanoscale DOX
delivery systems. For example:
Self-assembly
Cy 5.5
Review
Anionic domain
Extracellular pH 7.4
Ca2+ 2 mM
N+
KO3S
SO3K
Endocytosis
Nucleus
DOX release
CaP layer
dissolution
Figure 4. Shell-specific calcium phosphate mineralization of doxorubicin-loaded poly(ethylene glycol)-poly(l-aspartic acid)-poly(lphenylalanine) micelles and triggered release of doxorubicin by acidic intracellular compartments. CaP: Calcium phosphate; Cy:
Cyanine; DOX: Doxorubicin; PEG: Poly(ethylene glycol); PAsp: Poly( l-aspartic acid); PPhe: Poly(l-phenylalanine).
495

the outer surface (deshielding) at tumor extracellular pH have been developed to facilitate tumor cell
uptake without compromising their long circulation
time. This pH-triggered deshielding approach offers
an indispensable alternative to cell-specific active
targeting of solid tumors (based on, e.g., antibodies,
peptides and aptamers, among others);
Acid-sensitive nanoparticles that are prone to swelling, dissolution or degradation at endosomal/lysosomal pH (4.06.5) have been devised to obtain
fast intracellular DOX release in the tumor cells. To
further enhance their in vivo stability, acid-sensitive
nanoparticles can also be chemically cross-linked
(via either degradable or nondegradable cross-links);
pH-sensitive nanoparticles have been shown to facilitate disruption of endosomes via the proton sponge
effect and/or active interactions with endosomal
membranes (caused by deshielding, and low molecular
weight degradative products, among others);
pH-sensitive cross-linking of nanoparticles has
been utilized not only to enhance their in vivo stability, inhibit premature drug release, prolong drug
circulation time and improve drug accumulation at
the tumor site, but also to maintain fast drug release
inside target tumor cells due to cleavage of crosslinks under endosomal/lysosomal compartments;
Dual pH-sensitive nanoparticles (i.e., one sensitive
to tumor pH and the other to endosomal/lysosomal
pH) have been designed to accomplish prolonged
circulation time and efficient tumor cell uptake, as
well as fast intracellular drug release. pH-sensitive
nanoparticulate DOX formulations have demonstrated superior in vitro and in vivo antitumor
activity to free DOX and pH-insensitive controls.
Interestingly, several studies have shown that DOXloaded pH-sensitive nanoparticles are particularly
effective for the treatment of MDR cancers.
It should be noted that although not included in this
review, dual- and multi-stimuli-sensitive nanoparticles
that respond to pH and other stimuli (e.g., redox, enzyme
and near infrared, among others), either simultaneously
at the same location or in a sequential manner in different compartments, have been developed to offer unprecedented control over DOX delivery and release, leading
to superior in vitro and/or in vivo anticancer potency [74] .
In spite of significant progress in the development of
pH-sensitive nanoparticles for DOX delivery, most are
proof-of-concept studies and none have advanced to clinical evaluations. The first bottleneck with these smart
496
delivery systems, like other sophisticated anticancer

drug delivery systems, is that they are usually based on
innovative polymers that are difficult to obtain approval
for from the authorities for medical and pharmaceutical
uses. In the future, effort should be directed to advancement of pH-sensitive nanoparticles from well-accepted
biomedical materials such as biodegradable polyesters,
polycarbonates, polypeptides, PEG and dextran, among
others. It should be noted that DOX-loaded pH-sensitive nanoparticles, although displaying better in vitro
and in vivo antitumor activity as compared with free
DOX and pH-insensitive counterparts, are far from
optimal in terms of therapeutic performance. Given the
fact that pH differences between tumor tissues or endosomal/lysosomal compartments and normal tissues are
small, pH response is often found to be slow (ranging
from several hours to a few days), which results in only
partial DOX release at the tumor site or inside the tumor
cells. In order to achieve maximum therapeutic effects,
however, drugs should preferentially be dumped upon
arriving at the target site. To this end, nanoparticulate
DOX formulations with super pH sensitivity should
be developed for more effective cancer chemotherapy.
Moreover, it should be noted that the presence of multidrug resistance in cancer cells and cancer stem cells is
responsible for the failure of many clinical treatments
using either traditional drug formulations or nanomedicines [75] . Hence, it is of the utmost importance to
invent pH-sensitive nanoparticulate DOX formulations
that can reverse multidrug resistance in cancer cells and
effectively kill cancer stem cells. In the future, efforts
should be directed to the development of robust, tumortargeting and super pH-sensitive nanoparticles based
on well-established biocompatible materials for efficient
delivery and release of DOX into solid tumor cells, as
well as cancer stem cells. We are convinced that based
on pH-sensitive nanoparticles, innovative and smart
DOX nanomedicines may be developed for safe, efficient and targeted cancer therapy.
Financial & competing interests disclosure
This work is financially supported by research grants from
the National Natural Science Foundation of China (NSFC
51003070, 51103093, 51173126, 51273137 and 51273139),
the National Science Fund for Distinguished Young Scholars (NSFC 51225302) and a project funded by the Priority
Academic Program Development (PAPD) of Jiangsu Higher
Education Institutions. The authors have no other relevant
affiliations or financial involvement with any organization or
entity with a financial interest in or financial conflict with the
subject matter or materials discussed in the manuscript apart
from those disclosed.
No writing assistance was utilized in the production of this
manuscript.
Review
Executive summary
Tumor extracellular pH-sensitive nanoparticles
pH-responsive nanoparticles that reverse surface charge or expose ligands such as TAT and biotin at the
outer surface (deshielding) at tumor pH have been developed to facilitate tumor cell uptake, without
compromising their long circulation time.
Tumor pH-sensitive nanoparticles have been designed to achieve accelerated drug release at the tumor site.
Endosomal/lysosomal pH-sensitive nanoparticles

Acid-sensitive nanoparticles that are prone to swelling, dissolution or degradation at endosomal/lysosomal pH
have been devised to obtain fast intracellular doxorubicin release in the tumor cells.
pH-sensitive nanoparticles have been shown to facilitate disruption of endosomes via the proton sponge
effect and/or active interactions with endosomal membranes.
pH-sensitive cross-linking of nanoparticles has been utilized not only to enhance their in vivo stability and
improve drug accumulation at the tumor site, but also to maintain fast drug release inside target tumor cells.
Doxorubicin-loaded pH-sensitive nanoparticles are particularly effective for the treatment of multi-drug
resistant cancers.
Dual pH-sensitive nanoparticles

Dual pH-sensitive nanoparticles have been designed to accomplish a prolonged circulation time and efficient
tumor cell uptake, as well as fast intracellular drug release.
anticancer drug delivery: promises, progress and prospects.
Nano Today 7(5), 467480 (2012).
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Review
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orubicin

pH-sensitive polymeric nanoparticles for

10.2217/NNM.13.212 2014 Future Medicine Ltd

limits its dose and substitution for DOX HCl

Nanomedicine (2014) 9(3), 487499

Review Meng, Zhong, Cheng, Deng & Zhong

Nanomedicine (2014) 9(3)

future science group

Negative/neutral surface + Positive charge

or side chains. For example, van Hest et al. reported

future science group

sive hydrolysis behavior [21] . The imine linker, while

Review Meng, Zhong, Cheng, Deng & Zhong

Nanomedicine (2014) 9(3)

premature drug release as a result of covalent linking

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PEG-PTTMA-PAA (MnPEG > MnPAA)

pH-sensitive acetal degradation, swelling

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pH-activatable DOX prodrug nanogels by conjugating

Review Meng, Zhong, Cheng, Deng & Zhong

Nanomedicine (2014) 9(3)

pH 7.07.4, whereas they were disassembled at a pH of

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tumor targeting, which is an appealing alternative to

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bond and fully bloomed to release DOX at endosomal

Review Meng, Zhong, Cheng, Deng & Zhong

Acidic extracellular fluid

Nanomedicine (2014) 9(3)

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CH3 OCH2CH2 113NH COCHNH

Tumor extracellular pH-sensitive nanoparticles

Inorganic CaP layer

Conclusion & future perspective

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Review Meng, Zhong, Cheng, Deng & Zhong

Nanomedicine (2014) 9(3)

delivery systems, like other sophisticated anticancer

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Endosomal/lysosomal pH-sensitive nanoparticles

Dual pH-sensitive nanoparticles

Peer D, Karp JM, Hong S, Farokhzad OC, Margalit R,

Davis ME, Chen Z, Shin DM. Nanoparticle therapeutics:

Haag R, Kratz F. Polymer therapeutics: concepts and

Seymour LW, Ferry DR, Kerr DJ et al. Phase II studies of

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Jain RK, Stylianopoulos T. Delivering nanomedicine to solid

Lammers T, Kiessling F, Hennink WE, Storm G. Drug

Deng C, Jiang YJ, Cheng R, Meng FH, Zhong ZY.

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Presents the state of the field, as well as future challenges of

Rofstad EK, Mathiesen B, Kindem K, Galappathi K. Acidic

Grabe M, Oster G. Regulation of organelle acidity. J. Gen.

Dong D-W, Tong S-W, Qi X-R. Comparative studies of

Kim D, Gao ZG, Lee ES, Bae YH. In vivo evaluation of

Meng FH, Cheng R, Deng C, Zhong ZY. Intracellular drug

Qiu L, Zheng C, Zhao Q. Mechanisms of drug resistance

Huo M, Zou A, Yao C et al. Somatostatin receptor-mediated

Review Meng, Zhong, Cheng, Deng & Zhong

Brinkhuis RE, Visser TR, Rutjes F, van Hest JC. Shedding