Journal of Controlled Release 244 (2016) 108–121

Contents lists available at ScienceDirect

Journal of Controlled Release

journal homepage: www.elsevier.com/locate/jconrel

Review article

To exploit the tumor microenvironment: Since the EPR effect fails in the
clinic, what is the future of nanomedicine?
F. Danhier
Université catholique de Louvain, Louvain Drug Research Institute, Advanced Drug Delivery and Biomaterials, Avenue Mounier, 73 bte B1 73.12, 1200 Brussels, Belgium

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

Article history: Tumor targeting by nanomedicine-based therapeutics has emerged as a promising approach to overcome the
Received 19 September 2016 lack of specificity of conventional chemotherapeutic agents and to provide clinicians the ability to overcome
Received in revised form 26 October 2016 shortcomings of current cancer treatment. The major underlying mechanism of the design of nanomedicines
Accepted 7 November 2016
was the Enhanced Permeability and Retention (EPR) effect, considered as the “royal gate” in the drug delivery
Available online 18 November 2016
field. However, after the publication of thousands of research papers, the verdict has been handed down: the
Keywords:
EPR effect works in rodents but not in humans! Thus the basic rationale of the design and development of
EPR effect nanomedicines in cancer therapy is failing making it necessary to stop claiming efficacy gains via the EPR effect,
Tumor targeting while tumor targeting cannot be proved in the clinic. It is probably time to dethrone the EPR effect and to ask the
Nanomedicine question: what is the future of nanomedicines without the EPR effect? The aim of this review is to provide a gen-
Drug delivery eral overview on (i) the current state of the EPR effect, (ii) the future of nanomedicine and (iii) the strategies of
Cancer modulation of the tumor microenvironment to improve the delivery of nanomedicine.
© 2016 Elsevier B.V. All rights reserved.

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 109
2. Tumor targeting. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 109
2.1. Passive targeting: Background of the EPR effect . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 109
2.2. Active targeting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 109
2.3. Clinically used nanomedicines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 109
3. Heterogeneity in the EPR effect: a new clinically relevant EPR effect model . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 112
3.1. The heterogeneity or lack of fenestrations in the tumor endothelium . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 112
3.2. Hypoxic areas . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 112
3.3. The lower and heterogeneous pericyte coverage . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 112
3.4. Heterogeneous basement membrane . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 113
3.5. The higher and heterogeneous density of the ECM . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 113
4. What is the future for nanomedicines without EPR effect?. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 114
4.1. Toxicity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 114
4.2. Local delivery . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 114
4.3. Nanomedicines as a tool for imaging the tumor microenvironment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 114
5. Strategies to improve delivery . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 116
5.1. Vascular mediators involved in the EPR effect . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 116
5.2. Microenvironment modifications/modulations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 117
5.2.1. Direct permeability enhancement . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 117
5.2.2. Indirect permeability enhancement . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 117
5.3. Multifunctional nanomedicines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 119
5.3.1. Tumor-penetrating peptides. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 119
5.3.2. Stimuli-responsive nanomedicine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 119

E-mail address: fabienne.danhier@uclouvain.be.

http://dx.doi.org/10.1016/j.jconrel.2016.11.015
0168-3659/© 2016 Elsevier B.V. All rights reserved.
 F. Danhier / Journal of Controlled Release 244 (2016) 108–121
6. Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
1. Introduction
Tumor targeting by nanomedicine-based therapeutics has emerged
as a promising approach to overcome the lack of specificity of conven-
tional chemotherapeutic agents and to provide clinicians the ability to
overcome shortcomings of current cancer treatment [1–3]. Drug deliv-
ery systems can be used to specifically target tumors and improve the
therapeutic index and the pharmacokinetic profile of anticancer drugs.
Nanomedicines are submicrometer-sized carrier materials which in-
tend to improve the biodistribution of systemically administered che-
motherapeutic drugs. Nanoformulation aims at improving the balance
between the efficacy and the toxicity of systemic chemotherapeutic
agent interventions [4]. Among others, the main examples of
nanomedicine formulations are liposomes [5], micelles [6], nanoparti-
cles [7] and polymer-drug conjugates [8].
In 2010, we wrote a review called “to exploit the tumor microenvi-
ronment: passive and active tumor targeting of nanocarriers for anti-
cancer drug delivery” [9]. At this time, the particular tumor targeting
nanomedicines was in the spotlight since nanomedicine–based thera-
pies kept the promise to overcome the lack of specificity of conventional
chemotherapeutic agents. The major underlying mechanism of the de-
sign of nanomedicines was the Enhanced Permeability and Retention
(EPR) effect, considered as the “royal gate” in the drug delivery field
since drug-loaded nanocarriers could enter and accumulate inside a
tumor and reduce the side effects associated with conventional treat-
ments. This recycled principle of the “magic bullet” first imagined by
P. Ehrlich in 1906 was then really promising. After the publication of
thousands of research papers, the verdict has been handed down: the
EPR effect works in rodents but not in humans! Now the question is:
what is the future of nanomedicines?
In this review, I would like to highlight other possibilities that
nanomedicines can still offer in cancer therapy even if the basic concept
of EPR effect is compromised. Several reviews are available for each sec-
tions and it would go beyond the scope of this review to discuss these
issues in detail. Rather, the aim of this review is to propose a general
overview of the nanomedicine-related issues that could be addressed
and to discuss for which strategy or advantage nanomedicines are
promising. These strategies should be particularly taken into account
in the future and must be considered from their early conception to clin-
ical translation.
2. Tumor targeting
2.1. Passive targeting: Background of the EPR effect
The EPR effect was first termed by Maeda in 1986. The fundamental
features of EPR physiology are hyperpermeable tumor vasculature
allowing the enhanced permeability of large particles—including pro-
teins, macromolecules, liposomes, micelles and other soluble particles.
These were large enough to avoid renal clearance into the interstitial
space of the tumor combined with impaired lymphatic drainage limit-
ing the clearance of tumor particles and causing enhanced retention of
those same particles (Fig. 1A) [10]. Indeed, nanocarriers (20–200 nm
in diameter) can extravasate and accumulate within the interstitial
space of tumors by transiting through the much larger endothelial
pores (10 to 1000 nm in diameter). The absence of functional lymphatic
vessels in most tumors contributes to nanocarrier entrapment and re-
tention [11]. In addition, the lack of functional lymphatic vessels and
the vascular hyperpermeability inside tumors results in interstitial
109
119
119
119
hypertension. This uniformly elevated interstitial fluid pressure (IFP)
reduces convective transport, while the dense extracellular matrix hin-
ders diffusion [2].
In 1979, Maeda et al. reported the first synthesis of the anticancer
protein neocarzinostatin (NCS) conjugated with a polymer (styrene
maleic acid copolymer, SMA) which has been called SMANCS [12]. In
1986, SMANCS was found to accumulate to a greater degree than NCS
in tumor tissues. Later, other plasma proteins, larger than 40 kDa,
showed selective tumor accumulation. These findings led to generaliz-
ing the concept of the EPR effect [10]. Typically, the EPR effect was illus-
trated using an intravenous injection of the dye Evans blue, which binds
to plasma albumin, forming a macromolecule model. 24 h after injec-
tion, the dye was found in tumor tissue and not in normal tissues [13].
Scintigraphy of 67Ga complexed to transferrin (90 kDA) also provides
a demonstration of the EPR effect because of the preferential tumor ac-
cumulation of complexes [11].
2.2. Active targeting
On the contrary of passive drug targeting, active drug targeting relies
on the use on specific ligands (antibodies or peptides) that can be grafted
to the nanomedicine surface and specifically bind to overexpressed recep-
tors at the target site [4]. In the active targeting strategy, two cellular tar-
gets can be distinguished: (i) the targeting of cancer cells (overexpression
of transferrin, folate, Epidermal Growth Factor Receptor or Glycoproteins
receptors) and (ii) the targeting of the tumor endothelium (overexpres-
sion of the Vascular Endothelial Growth Factors (VEGF), αvβ3 integrins,
the Vascular Cell Adhesion Molecule-1 (VCAM-1) or matrix metallopro-
teinases (MMP's) (Fig. 1B). Interestingly, some targets can be
overexpressed in both endothelial and cancer cells [9]. The active
targeting strategy promised to target the cancer tissue more specifically
than with the EPR effect alone. We know now that actively targeted nano-
particles are internalized via the receptor-mediated endocytosis. The in-
creased efficacy of these systems is thus due to an improved target cell
recognition and target cell uptake rather than to a better tumor accumu-
lation [4,14].
2.3. Clinically used nanomedicines
As demonstrated by the significant nanomedicine market, the applica-
tion of nanotechnology in the field of medicine has the potential to im-
prove the treatment of cancer [16] and several nanomedicines have
been approved by the US Food and Drug Administration (FDA) and Euro-
pean Medicines Agencies (EMA) for cancer therapy [17]. Table 1 shows an
exhaustive list of nanomedicines for cancer treatment, which are either
approved or in clinical trials. In this table, nanomedicines are classified
by nanocarrier (micelles, nanoparticles, polymer drug conjugate and lipo-
somes). The description of all these systems is beyond the scope of this re-
view, however, some nanomedicines are discussed hereunder.
The growing presence of novel nanomedicines raises the question of
whether approved nanomedicines really form nanoparticles; i.e. are im-
provements we observed from nanomedicines structure-related or do
they result from improved formulations? Do we even have sufficient
data to address this question? Regarding the large number of
nanomedicines on the market and in clinical trials, we cannot expect
to find a “one size fits all” answer.
The first nanotechnology drug delivery systems, described in the
1960s, were lipid vesicles, which were later called liposomes [18,19].
Subsequently, a variety of drug delivery nanosystems were developed
110 F. Danhier / Journal of Controlled Release 244 (2016) 108–121
Fig. 1. (A) Schematic representation of the conceptual passive targeting (EPR effect) of nanomedicine. (B) Active targeting of nanomedicine grafted with peptide or antibody able to bind
specific receptors overexpressed by (1) cancer cells or (2) endothelial cells.
Adapted from [9,15].
and, in 1976, the first sustained release drug delivery system was de-
scribed [20]. In 1980, the first example of the specific targeting of lipo-
some was described with a system able to respond to changes in pH
to trigger drug release [21]. In 1987, the first long-circulating liposome
was described, introducing the concept of PEGylation, where the lipo-
some surface was covered with polyethylene glycol (PEG) chains reduc-
ing opsonization and premature clearance, therefore increasing blood
circulation times and potentially enhancing tumor accumulation [18].
In 1995, doxorubicin-loaded PEGylated liposomes (named Doxil® in
the USA and Caelyx® in other countries) were approved for the treat-
ment of AIDS-associated Kaposi's sarcoma (Table 1) [22]. Doxil® was
the first drug carrier to reach the market in the United States, gaining
FDA approval only five years after its development was first reported
[22]. The clinical therapeutic efficacy of Doxil® was superior in ovarian
cancers and AIDS-related Kaposi's sarcoma compared to standard ther-
apies, while equivalent in multiple myeloma and metastatic breast can-
cer [23,24]. However, the main benefit of Doxil® in treating solid tumors
was the reduced cardiotoxicity gained by limiting doxorubicin exposure
to cardiac tissue. However, toxicity was not reduced across the board,
but was aggravated in other categories, most notably skin toxicity in
the form of palmar–plantar erythrodysesthesia (PPE)—hand and foot
syndrome. This toxicity directly results from the extended circulation
time of the PEGylated liposomes, which allows time for the kinetically
slow process of extravasation into the skin to become significant.
Doxil® formulation failed to show drastic enhanced tumor accumula-
tion over free doxorubicin, indicating that a minimum EPR is unreliable
in human tumors. The EPR hypothesis would suggest that longer circu-
lation times produce higher intratumoral accumulation, which should
in turn improve patient outcomes. It could mean that clinical EPR is
not as well understood as originally thought [24].
The initial approval of Doxil® was followed by various other liposo-
mal formulations including, (i) Ambisome® for the delivery of
amphotericin B for infectious disease indications, (ii) DaunoXome®,
50 nm daunorubicin liposomes approved for AIDS-associated Kaposi's
sarcoma, (iii) Myocet®, a 150 nm non-PEGylated liposomal doxorubicin
formulation approved in Europe and Canada for metastatic breast can-
cers, and (iv) MM-398®, a 100 nm liposomal formulation of irinotecan
recently approved for the treatment of pancreatic adenocarcinoma in
combination with 5-fluorouracil (5-FU) and leucovorin (Table 1).
While liposomes are a dominant class of nanomedicine, other
nanomedicines have been approved, such as nanoparticles and micelles
for cancer therapy. These nanoformulations include Abraxane®, 130 nm
albumin-bound paclitaxel particles, indicated for metastatic breast can-
cer and recently for pancreatic adenocarcinoma. Outside of the global
market, Genexol-PM® is a 20–50 nm polymeric micelle formulation of
paclitaxel approved in South Korea for metastatic breast cancer (Table
1) [16].
Abraxane®, with an annual revenue estimated at $967 million, is one
of the major success stories in the development of nanomedicines.
Abraxane® (an albumin-bound nanoparticle of paclitaxel) was devel-
oped to retain the therapeutic benefits of paclitaxel but eliminate the
toxicities associated with the emulsifier Cremophor® EL in the commer-
cialized paclitaxel formulation (Taxol®) [25]. The maximum tolerated
dose (MTD) of Abraxane® was approximately 50% higher than reported
for Taxol®. Pharmacokinetic assessments also showed that paclitaxel
clearance and volume of distribution were higher for Abraxane® than
for Taxol®. These differences in pharmacokinetic properties may be as-
sociated with the higher intratumoral concentrations observed with
Abraxane® compared with the equivalent dose of Taxol®. This improve-
ment was credited to the receptor-ligand targeting via active albumin
transport pathways. It is difficult to decipher the exact impact of the
EPR effect, however, the increase in efficacy could be easily explained
by the increased dose of available drug alone [24]. It should be noted
that it is unclear whether or not the nanoparticles dissolved when in-
fused into patients. Thus, the clinical benefit from Abraxane® is probably
not due to its functioning as nanoparticles but to other factors such as
the removal of Cremophor® EL from the formulation that causes toxic-
ities of its own [14,26].
 F. Danhier / Journal of Controlled Release 244 (2016) 108–121 111

Table 1
Examples of clinically used nanomedicines in cancer therapy.
Clinical data are extracted from http://www.clinicaltrials.gov (2016).

Nanocarriers Drug Name Indications Status

Micelles
Paclitaxel Genexol®-PM Breast, lung, pancreatic cancer Approveda
Recurrent breast cancer
Paclitaxel Paclical Ovarian cancer III
Paclitaxel NK105 Gastric cancer III
Doxorubicin NK911 Various solid tumors II
Cisplatin NC-6004 Pancreatic cancer I/II
Epirubicin NC-6300 Various solid tumors I
Oxaliplatin NC-4016 Various solid tumors I

Nanoparticles
Albumin- paclitaxel Abraxane® Metastatic breast cancer Approved
Doxorubicin Transdrug® Hepatocarcinoma Approved
Doxorubicin BA-003 Hepatocellular carcinoma III
Mitoxantrone DHAD-PBCA-NPs Hepatocellular carcinoma II
e
Docetaxel BIND-014 NSCLCb II
Camptothecin CRLX101 NSCLCb II
Camptothecin IT-101 Solid tumors I/II
dnG1 plasmid DNA Rexin-G Various solid tumors I/II
Anti-PKNe siRNA Atu027 Various solid tumors I/II
Docetaxel ABI-008 Breast, prostate cancers I/II
Rapamycin ABI-009 Non-hematologic malignancies I/II
BikDD plasmid DNA C-Visa-BikDD Pancreatic cancer I
Paclitaxel Nanoxel® Advanced breast cancer I
Docetaxel Docetaxel-PNP Various solid tumors I
e
Anti-RRM2 siRNA CALAA-01 Various solid tumors I

Polymer drug conjugate

Asparginase Oncaspar® Leukemia III
Paclitaxel Xyotax® Breast, ovarian cancer III
(CT-2103) Advanced lung cancer III
Paclitaxel Taxoprexin® Various solid tumors II-III
Doxorubicin PK1 Breast, lung, colon II
IFN-α2a/b PegAsys/PegIntron® Melanoma/Leukemia II
Oxaliplatin ProLindac® Ovarian cancer II
Diaminocyclohexane platinium AP 5346 Ovarian cancer II
Docetaxel DEP® Advanced cancers I
CPT XMT-1001 Gastric, NSCLC cancers I

Liposomes
Doxorubicin Doxil/Caelix® Ovarian, metastatic breast cancer, Kaposi sarcoma Approved
Doxorubicin Myocet® Breast cancer Approved
Daunorubicin DaunoXome® Kaposi Sarcoma Approved
Vincristine Onco-TCS® Non-hodgkin lymphoma Approved
Cytabirine Depocyt® Lymphomatus meningitis Approved
Vincristine Marqibo® Leukemia Approved
Mifamurtide Mepact® Osteosarcoma Approved
Irinotecan Onivyde® (MM-398) Metastatic pancreas cancer Approved
Paclitaxel Lipusu® NSCLC, breast cancer Approvedc
Paclitaxel PICN® Metastatic breast cancer Approvedd
Doxorubicin Thermodox® Liver, breast cancers III
Cisplatin Lipolatin NSCLC III
9-nitrocamptothecin 9NC-LP Hepatocellular carcinoma II/III
Cisplatin SPI-077 Solid tumors I/II/III
Oxaliplatin Lipoxal Advanced cancers II
Paclitaxel EndoTAG-1 Breast, liver, pancreatic cancers II
Lutotecan OSI-211 Solid cancers II
Docetaxel LE-DT Pancreatic, prostatic cancers I/II
Paclitaxel LEP-ETU Breast cancer, leukemia I/II
PLK1 siRNA TKM-080301 Neuroendocrine tumors I/II
PKN3 siRNA Atu027 Pancreatic cancer I/II
Doxorubicin 2B3-101 Solid tumors I/II
Irinotecan NL CPT-11 Reccurent high grade glioma I
CEBPA saRNA MTL-CEBPA Liver cancer I
Topotecan TL1 Various solid tumors I
Irinotecan IHL-305 Advanced solid tumors I
Docetaxel ATI-1123 Solid tumors I
Vinorelbine Alocrest Bresat and lung cancers I
Cisplatin LiPlaCis Advanced solid tumors I
e
Doxorubicin MCC-465 Metastatic stomach cancer I
e
p53 gene SGT-53 Various solid tumors I
a
Approved in Korea.
b
Advanced Non-Small Cell Lung Cancer.
c
Approved in China.
d
Approved in India.
e
Targeted nanomedicine.
112 F. Danhier / Journal of Controlled Release 244 (2016) 108–121
For more than thirty years, great effort has been expended to devel-
op better nanocarriers. The advantages of nanomedicines were myriad;
they could be endlessly manipulated to take on many shapes, sizes and
functionalities; they could simultaneously perform multiple tasks, such
as treatment and imaging; they could carry large quantities of therapeu-
tic agents, and they would automatically preferentially accumulate in
tumors by virtue of their size while sparing the rest of the body. In
spite of these advantages, of the hundreds of drug carrier designs re-
ported, only few have been approved for clinical use. The two most suc-
cessful formulations, Doxil® and Abraxane®, are surprising due to their
simplicity.
The identification of new disease biomarkers is essential in the
choice and development of targeting ligands. While non-targeted
nanomedicines have been clinically efficacious, numerous contradicting
data regarding the benefit of adding a targeting ligand have been shown
[27]. The tumor accumulation is more largely mediated by physico-
chemical properties of the nanoparticles compared to active targeting
[28]. Therefore, even in the absence of targeting ligands, nanomedicines
can be designed to better target a specific tissue, or be nonspecifically
absorbed by cells, by optimizing their physico-chemical properties
[29]. However, as aforementioned, specific uptake by cancer cells is fa-
cilitated by active targeting. Despite a large number of publications in
this area, only a few systems with active targeting are under investiga-
tion in clinical trials (Table 1) [30–32]. These targeted systems include
(i) MCC-465, doxorubicin encapsulated in immunoliposomes tagged
with the F(ab′)2 fragment of human monoclonal antibody GAH, which
binds to neoplastic stomach tissues [33]; (ii) SGT-53, a liposomal
nanocomplex designed for systemic, tumor-targeting delivery of the
p53 gene. In this nanodelivery system, an anti-transferrin receptor sin-
gle-chain antibody fragment (scFv) serves as the targeting moiety to
target cancer cells through the transferrin glycoprotein receptor [27];
(iii) BIND-014, docetaxel-loaded nanoparticles targeting the Prostate-
Specific Membrane Antigen (PSMA); and (iv) CALAA-01, siRNA-loaded
nanoparticles containing human transferrin as targeting ligand for bind-
ing transferrin receptors [34].
3. Heterogeneity in the EPR effect: a new clinically relevant EPR ef-
fect model
While the number of publications citing “EPR” and “nanomedicine”
has exponentially increased, this creative flourish has largely failed to
translate into the clinic. While the EPR effect has been well documented
in small animal models, clinical data is less clear. Nanomedicines have
almost uniformly failed to produce improved efficacy in the clinic, in
spite of tremendously successful preclinical results (see examples of
Abraxane® and Doxil® in the Section 2.3) [24,35].
Models being used to study EPR in laboratories are not sufficiently
accurate and require reconsideration. Indeed, murine tumor models
drastically differ from human cancers in many aspects including the
rate of development, the size relative to host, metabolic rates and host
lifespan. More particularly, human tumors present many differences in
their tumor microenvironment compared with murine tumors simply
because most rodent tumors grow much faster. A subcutaneous tumor
grows to ±1 cm (≈0.5 g) within 2–4 weeks. In human, this would rep-
resent a ± 20 cm (≈ 1–2 kg) growth [4]. The large tumor-to-body
weight ratio in mice compared to human patients may significantly
alter the pharmacokinetics of drug carriers. Murine tumors are often ob-
served to be as much as 10% of the mouse's body weight. The volume of
an equivalent tumor in a 70 kg human would be the size of a basketball
[36]. Such large tumors filter a significant proportion of the injected
dose of a drug and act as a repository for the drug, effectively accentuat-
ing the efficacy while mitigating the toxicity. Moreover, tumor microen-
vironment in human cancers present some major differences in
comparison with murine tumors: (i) the heterogeneity or lack of fenes-
trations in the tumor endothelium, (ii) the heterogeneity of blood flow
through tissues, leading to regions becoming acidic or hypoxic [37], (iii)
the lower and heterogeneous pericyte coverage, (iv) the heterogeneous
basement membrane (BM) and (v) the higher and heterogeneous den-
sity of the extracellular matrix (ECM), leading to an elevated IFP which
forces nanomedicines to escape the blood compartment by diffusion
rather than the much more efficient convective transport [37–39].
Hence, regarding these notable differences in the tumor microenviron-
ment, a new schematic representation of the passive targeting (EPR ef-
fect) in human tumors is illustrated in Fig. 2. The practicalities of
evaluating the EPR effect in human tumors are relatively costly and
time-consuming. However, biological conditions driving EPR variability
is crucial. Tumor histology is not sufficient to predict EPR effect. Imaging
modalities (such as computer tomography, magnetic resonance imag-
ing (MRI) or functional ultrasound) are needed to investigate the corre-
lation between vascularization (permeability, perfusion, IFP) and intra-
tumor accumulation. Alternatively, researchers could use more relevant
in vivo tumor models, such as patient-derived tumor explant (PDX)
models, which more faithfully reflect the morphology, complexity and
heterogeneity of clinical tumors. Indeed, these tumor models are more
mature and less permeable than xenografts [40].
3.1. The heterogeneity or lack of fenestrations in the tumor endothelium
Because of the rapid murine tumor growth, blood vessels in mouse
tumor generally do not develop properly, and they consequently tend
to be much more leaky with endothelial cells presenting many fenestra-
tions [4]. Unfortunately, in humans, not all tumor vessels are leaky,
which causes a heterogeneous distribution of pore sizes and thus, het-
erogeneous extravasation and delivery [2].
An illustration of this statement consists in the difference of tumor
accumulation of 5 nm-sized radiolabeled HPMA copolymers in two dif-
ferent rat tumor models. On one side, in Dunning AT1 prostate carcino-
ma growing to 1 cm in diameter within 2 weeks, the preferential
accumulation of copolymers in tumor tissue has been correlated to
poorly differentiated blood vessels and hardly any coverage with
pericytes. On the other hand, in Dunning H prostate carcinoma growing
to 1 cm in diameter in 1 year, a lesser accumulation of copolymers in
tumor tissue has been correlated to properly differentiated blood ves-
sels and dense coverage with pericytes [41].
3.2. Hypoxic areas
The heterogeneity of blood flow through tissues leads to regions be-
coming acidic or hypoxic: most human solid tumors contain hypoxic
areas. Hypoxia is the direct consequence not only of the chaotic prolifer-
ation of cancer cells that places them at some distance from the nearest
capillary, but also of the abnormal structure of the neovasculature net-
work resulting in transient blood flow [42]. The subsequent hypoxia in
tumor cells not only induces resistance to radiotherapy, but also causes
resistance to several cytotoxic drugs, while also inducing genetic insta-
bility and selecting for more malignant tumor cells with potentially
metastatic properties. Additionally, another consequence of the hetero-
geneity in human cancer relating to the abnormal coagulation or
clotting in tumor vessels is the resulting fibrinolysis or thrombocytope-
nia that can affect the EPR effect [43].
3.3. The lower and heterogeneous pericyte coverage
The relationship between pericyte coverage and nanomedicine ex-
travasation has been investigated in detail. In murine models, all
tumor microvessels have low and loose pericyte coverage. In human tu-
mors, emerging evidence has demonstrated heterogeneous pericyte
coverage within one single tumor. By defining pericytes as alpha-
smooth muscle actin (α-SMA) positive cells attached to endothelial
cells, malignant tumors have been classified into high pericyte coverage
and low-pericyte coverage subtypes. Additionally, more coverage has
been related to a worse prognosis and more fibrotic interstitium for
 F. Danhier / Journal of Controlled Release 244 (2016) 108–121
Fig. 2. Schematic representation of the passive targeting of nanomedicine (EPR effect) in human tumors. Tumor microenvironment in human cancers present some major differences in
comparison with murine tumors: (1) the heterogeneity or lack of fenestrations in the tumor endothelium, (2) the heterogeneity of blood flow through tissues, leading to regions becoming
acidic or hypoxic, (3) the lower and heterogeneous pericyte coverage, (4) the heterogeneous basement membrane and (5) the higher and heterogeneous density of the extracellular
matrix (ECM), leading to an elevated interstitial fluid pressure (IFP).
Adapted from [15].
different tumors (pancreas, gastric, renal cancers or glioblastoma; 60–
70% coverage), when compared to low-coverage cancers with a better
prognosis such as colon and ovarian cancers (10% coverage) [44,45].
Disparate intratumoral nanoparticle transport in response to cytokine-
mediated modification of pericyte coverage has been observed in two
types of murine tumors. Murine colon cancer CT26 is an example of
low-pericyte coverage tumor, while BxPC3 pancreatic model is
hypovascularized with pericyte coverage of 70%. Kinase inhibitors
were able to improve 2 MDa dextran and Doxil® penetration, leading
to increased tumor inhibition, in the BxPC3 tumor model. This observa-
tion is due to the blockage of pericyte proliferation induced by kinase in-
hibitors. On the CT26 tumor model, the pericyte coverage was too low to
achieve any additional effect with the kinase inhibitors [46,47]. The re-
lationship between pericyte coverage and nanoparticle extravasation
depends on the original pericyte coverage, blood vessel stabilization
and extracellular content, since pericyte is an indispensable factor for
vessel stabilization and maturation. Neither leaky, unmatured blood
vessels with little coverage, nor over-matured vessels with abundant
pericyte coverage are suitable for nanomedicine delivery [45].
3.4. Heterogeneous basement membrane
The BM is a specialized form of the ECM that serves as a scaffold for
endothelial and mural cells. The main components of the BM are lami-
nin and type IV collagen, which form distinct sheet-like structure linked
together by nidogen and heparin sulfates [45]. Heterogeneous BM mor-
phologies exist in different regions of the same tumor or different tumor
types. Even if BM does not induce elevated IFP, the presence of BM limits
the penetration of nanomedicines through pole-opening on capillary
walls such as the ECM (see the next section). Hence, diffusion of
nanomedicines with sizes larger than 100 nm could be severely affected
113
by mesh size and thickness of the BM. Therefore, the type IV collagen
density, mesh size, number of layers, and association with the vessels
form a barrier to the nanomedicine extravasation [45,48,49].
Extravasation of 1 nm doxorubicin, 50 nm macromolecule FITC-
tagged dextran and 80 nm PEGylated liposomes were evaluated on
the BM/vessel over-lapped 3LL model and the BM/vessel dissociated
model 4T1. The extravasation pattern of small molecules (including
doxorubicin and dextran) was comparable, suggesting that vascular col-
lagen could only modulate the transport of small molecules to a limited
extent. However, the extravasation of liposomes was significantly dif-
ferent between these two types of tumors. Extravasation only occurred
from the vessels that were not tightly covered by collagen type IV [50].
3.5. The higher and heterogeneous density of the ECM
Elevated intratumoral IFP has been well documented not only in an-
imals but also in clinical tumors [1]. A direct consequence of the elevat-
ed IFP levels is that the main mechanism of mass transport across the
vessel wall is diffusion, a process that is much slower than convection.
The large size of nanoparticles along with the uniformly elevated IFP
in tumors hinder transport across the vessel walls and compromise
the benefits of the EPR effect [2]. Additionally, the diffusion coefficient
of nanoparticles is considerably hindered by interactions with the ECM.
The ECM consists in a cross-linked network of collagen and elastin fi-
bers, glycoproteins, proteoglycans and hyaluronic acid. Contrarily to
BM, the ECM mainly consists in a network of collagen (Types I, II, III)
interacting with the other components. The ECM not only provides
structural integrity, but also helps to transport important nutrients
and oxygen to support cell growth [51]. At the cellular level, ECM is lo-
calized at two different sites, in the basement membrane and in the ma-
trix of the interstitial space [52]. Cancer-associated fibroblasts (CAFs)
114 F. Danhier / Journal of Controlled Release 244 (2016) 108–121
are the major players involved in the ECM-mediated malignant changes,
which include upregulated ECM synthesis, posttranslational modifica-
tions and matrix metalloproteinase (MMP)-induced ECM remodeling
[52,53]. Diffusion of nanoparticles across the thick interstitial matrix is
the last step to approaching tumor cells before cell internalization. For
tumors with less ECM, nanomedicine can easily diffuse. However, for tu-
mors with a thick interstitial matrix, this process is more intractable.
Unfortunately, the ECM is much more dense and thick in human tumors
than in murine ones [45]. Collagen becomes increasingly important as
the particle size approaches or exceeds the matrix mesh size that ranges
between 20 and 40 nm in solid tumors. Particles larger than the mesh
size can be prevented from diffusing from the matrix entirely, those
near the mesh size can be hindered, and small particles can pass
through relatively uninhibited [36]. In conclusion, the dense and hetero-
geneous structure of the ECM combined with the subsequent elevated
IFP in human tumors blocks large nanomedicine penetration and results
in the low and heterogeneous distribution of nanomedicine into
tumors.
4. What is the future for nanomedicines without EPR effect?
EPR effect is probably the most important concept in new anticancer
drug delivery systems. Nevertheless, as aforementioned, the EPR effect
is really heterogeneous in humans. Thus the basic rationale of the design
and development of nanomedicines in cancer therapy is failing and it
would be necessary to stop claiming efficacy gains via the EPR effect,
while the tumor targeting cannot be proved in the clinic. It is probably
time to dethrone the EPR effect and to ask the question: what is the fu-
ture of nanomedicines without EPR effect?
In this section, I would like to highlight the different advantages and
possibilities that nanomedicines can offer in cancer therapy. Three main
applications of nanomedicines do not require the EPR effect: (i) the de-
crease of toxicity of chemotherapeutic agents, (ii) the local delivery of
anticancer agents and (iii) the use of nanomedicines as a tool for imag-
ing the tumor microenvironment.
4.1. Toxicity
Regarding the two major examples of nanomedicines on the market
(Abraxane® and Doxil®), nanomedicines are able to reduce the toxicity
of chemotherapeutic agents, rather than to improve their efficacy. Most
conventional chemotherapies are effective but their clinical use is limit-
ed by their toxicity (neuropathy induced by paclitaxel; cardiotoxicity
induced by doxorubicin or 5-fluorouracil, etc.). Drug delivery systems
able to reduce side effects while increasing the maximum tolerated
dose (MTD) would be in itself beneficial for patients. For example, the
MTD of Abraxane® was approximately 70–80% higher than that report-
ed for Taxol®. In a Phase III study of 454 patients with metastatic breast
cancer given Abraxane® or Taxol® intravenously every 3 weeks, re-
sponse rates were significantly greater in patients treated with
Abraxane® than those receiving Taxol®. Pharmacokinetic assessment
also showed that paclitaxel clearance and volume of distribution were
higher for Abraxane® than for Taxol®. Clearance was 13 L per hour per
m2 for Abraxane® versus 14.76 L per hour per m2 for Taxol® (p =
0.048). There are numerous examples of highly effective combination
of nanomedicine with anticancer agents [54]. Nevertheless, the possibil-
ities of combinations are limited by the toxicity of each product. To se-
lect a potent effective combination, the two drugs need (i) to present
different mechanisms of action, overcoming the problems connected
with tumor cell heterogeneity and their implication for drug resistance;
and (ii) to induce different side effects, avoiding cumulative toxicity.
Since nanomedicine is able to decrease side effects, it can be used in
combination with conventional chemotherapy, opening the way for
other combinations. Abraxane® has been combined very well with
bevacizumab and with gemcitabine in patients suffering from metasta-
tic breast cancer, as was Genexol®-PM with cisplatin and with
carboplatin. Doxil® has also been combined very well with the
alkylating agent canfosfamide in ovarian carcinoma and with the pro-
teasome inhibitor bortezimab in multiple myeloma [4].
4.2. Local delivery
By definition, the local delivery does not require EPR effect. Local
chemotherapy is of great importance in cases of solid tumors, which
represents N 85% of human cancers. The local delivery of anticancer
drugs is particularly preferred if cancer cells are accumulated in parts
of the body which are easily accessible from the outside, such as the
skin (melanoma). In this case, topical iontophoretic or transdermal ad-
ministrations are chosen. When the tumor site is not located superficial-
ly, different regional drug delivery strategies can be used, including the
injection of anticancer agents directly into tumor tissue, the use of in-
jectable in situ forming drug carriers or injectable platforms. These
methods can be successfully applied in the treatment of solid tumors lo-
cated in breast, lungs, liver or bones. Higher achievable drug concentra-
tions combined with minimal side effects are the two main advantages
of this kind of therapy [54,55]. Moreover, the use of nanomedicine al-
lows the sustained release of the drug and thereby enhances the efficacy
of the treatment. Nevertheless, the current challenge of the develop-
ment of local drug delivery systems consists in the enhancement of
drug stability and the limitation of drug diffusion away from the
tumor. A particularly interesting indication of local drug delivery sys-
tems is glioblastoma (GBM), since conventional chemotherapy fails
due to the poor crossing of the blood-brain barrier (BBB). Many strate-
gies have been developed to circumvent the BBB and reach therapeutic
concentrations of chemotherapeutic drugs in brain tumors. Among
them, small lipophilic drugs have been used to passively pass the BBB,
while active compounds have been modified or incorporated into
nanocarriers in order to reach the brain parenchyma by passive
targeting or active targeting of the BBB endothelial cells [56]. Others
have tried to modify the BBB permeability or used focused ultrasounds
to transiently open the BBB for drug delivery [57]. Recently, a specific
and innovative hydrogel uniquely formed of lipid nanocapsules (LNC)
and lauroyl-gemcitabine (GemC12) has been developed and suggested
as local treatment for GBM. This formulation fulfills the definition of
both nanomedicine and hydrogel. Indeed, the formation of the hydrogel
is due to the location of the GemC12 at the oil-water interface of the LNC,
which allows the formation of H-bond cross-links between the drug
moieties and the immobilization of the water phase [58].
4.3. Nanomedicines as a tool for imaging the tumor microenvironment
Nanoparticles are frequently suggested as diagnostic agents. Never-
theless, none of these nanodiagnostics were finally approved for clinical
use. Indeed, the long-circulating properties of these nanoparticles,
allowing peaks ranging from 12 and 48 h, were unable to achieve the
rapid and highly site-specific contrast enhancement needed for an ef-
fective diagnosis [59]. The visualization of blood vessels is of high cancer
diagnostic relevance. As positive MR contrast agents, small paramagnet-
ic iron oxide (SPIO) nanoparticles were successfully applied in preclini-
cal and clinical trials but no product was finally approved for clinical use.
The only exception consists in the ultrasmall paramagnetic iron oxides
(USPIO) nanoparticles such as Sinerem®, Feridex®, Feraheme® and
Supravist®, which are used in the clinic for “vessel size imaging” only
in one particular application: mononuclear phagocytic system (MPS)
imaging. The MPS consists of liver and spleen, which are the main or-
gans in which nanoparticles are taken up and accumulate after intrave-
nous administration. For more details about the MPS imaging using
nanoparticles, readers are invited to read the review of Kiessling et al.
[59].
Contrast agents and therapeutic agents can be combined within a
single multifunctional nanomedicine, know as “theranostic
nanomedicine or nanotheranostics”. To be efficient, nanotheranostics
 F. Danhier / Journal of Controlled Release 244 (2016) 108–121 115

need to (i) selectively accumulate within the tumor tissue and (ii) to de-
liver therapeutic dose of therapeutic agents with a detectable dose of
contrast agent allowing detection of tumors at their earliest stage. The
applications of nanotheranostics are various: (i) the non-invasive as-
sessment of the nanomedicine biodistribution and target site accumula-
tion, (ii) the drug release monitoring, (iii) the triggered release of the
drugs and contract agents via an external stimulus and (iv) the predic-
tion of the therapeutic response, also called “personalized medicine”
[60]. All these approaches are extensively reviewed in the literature
[60–64]. In Fig. 3, the clinical relevance of each application of
theranostics is proposed. Among these applications, some could have
a potential impact for clinical use or on the contrary some could be
used in preclinical models as a tool to visualize the tumor microenviron-
ment allowing a better design of nanoparticles. Researchers developing
nanotheranostics often perform the in vivo non-invasive assessment of
the nanomedicine biodistribution and the drug release monitoring to
demonstrate the promising potential of their systems (Fig. 3A). I believe
that clinicians and pharmaceutical companies aiming at commercializ-
ing nanomedicines should in parallel develop nanotheranostics as a
tool for earliest phases of clinical trials since the initial biodistribution
and target site of accumulation, as well as the monitoring of the tumor
response, should be correlated. Nanotheranostics should therefore
allow a better understanding of how nanoparticles work. Alternatively,
in the specific case of nanotheranostics containing SPIO, it is possible to
guide SPIO-based theranostics directly to the target tumor tissue with
an external magnetic field, since SPIO presents superparamagnetic
properties (Fig. 3B). When a permanent magnet is applied externally
near the tumor region, the magnetic gradient produced exerts attractive
forces on magnetic nanoparticles delivered via the circulation [60]. This
targeting strategy could compensate the poor EPR effect of most tumors.
For example, Schleich et al. both quantify and visualize the accumula-
tion of multifunctional nanoparticles into the tumors by Electron Spin
Resonance spectroscopy and MRI, respectively. It has been
demonstrated that the combination of both active strategy and magnet-
ic targeting drastically enhanced (i) nanoparticle accumulation into the
tumor tissue with an 8-fold increase compared to passive targeting
(1.12% and 0.135% of the injected dose, respectively), (ii) contrast in
MRI and (iii) anti-cancer efficacy with a median survival time of
22 days compared to 13 for the passive targeting [60]. However, the suc-
cess of this strategy is limited by inadequate magnetic gradients. Many
strategies have been developed to overcome this problem [65–68]. An-
other prospect for imaging-guided drug delivery is for the prediction of
the therapeutic response also called “personalized medicine” (Fig. 3C).
For this concept, Lammers et al. discriminate two different approaches:
(i) the “biomarker approach” offers the opportunity to assist treatment
selection, response prediction and treatment monitoring; and (ii) the
“image guidance approach” allows planning, pre-operative guidance
and follow up of the therapeutic action [62]. Few clinical studies using
nanotheranostics have provided proof-of-principle for personalized
medicine. However, convincing clinical data are only available on tu-
mors associated with a strong EPR effect, such as Kaposi sarcoma [59].
For example, in a cohort of 7 patients, scintigraphic imaging with
Caelyx®-99mTc-DTPA showed an increased (2.8 times higher)
intratumoral drug accumulation compared to the surrounding healthy
tissue [69]. Hence, we can again legitimately question the clinical rele-
vance of this kind of system in the treatment of solid tumors. In this con-
text, via their non-invasive biodistribution visualization and the
importance of the EPR, theranostics should address this shortcoming.
Assuming that there will be a clear correlation between tumor concen-
tration and therapeutic efficacy, it will be highly important to properly
differentiate between low and high levels of target site accumulation.
These studies should also guide the specific use of nanomedicines in
clinical applications [60]. However, I believe that future clinical oncolo-
gy needs to take advantage of these theranostic tools to (i) understand
the fate of nanoparticles distributed in the tumor microenvironment
and to (ii) adapt the treatment to each specific case and find the best

Fig. 3. Clinical relevance of the three main applications of nanotheranostics in cancer treatment. (A) The non-invasive assessment of the nanomedicine biodistribution and the drug release
monitoring. (B) Magnetic theranostics directly target tumor tissue with an external magnetic field. (C) The prediction of the therapeutic response also called “personalized medicine”
which is divided into two different approaches: (i) the “biomarker approach” and (ii) the “image guidance approach”.
Adapted from [68] for panel A and from [62] for panel C.
116 F. Danhier / Journal of Controlled Release 244 (2016) 108–121
treatment for each patient. To further promote the clinical translation of
nanotheranostics for diagnostic and therapeutic purposes, an increased
interdisciplinary collaboration and knowledge exchange between sci-
entists from various disciplines is needed [62].
5. Strategies to improve delivery
Since recent data supports a considerable barrier role of tumors
resulting in imperfect or inefficient EPR effect, many researchers are
working to either increase the EPR effect or mitigate anti-EPR character-
istics [52]. Strategies to modulate or to counterattack the abnormal
tumor microenvironment have shown promising results in the en-
hancement of anticancer drug delivery systems, providing therefore a
second wind to nanomedicines in cancer therapy. A better understand-
ing of the tumor microenvironment may allow researchers to identify
the abnormal physiological processes that can be modulated to improve
drug penetration and efficacy in human solid tumors. Regarding formu-
lations currently in clinical use, nanoparticles with neutral charge and a
size range of 12–50 nm would be ideal as far as transport is concerned.
Micellar polymeric particles (i.e. NC-6004, NC-4016, NC-6300 and
Genexol-PM®) as well as Abraxane® and CRLX101 reside within this
range. Liposomal formulations usually have a larger size in the range
of 100 nm (e.g. Doxil®, MM-398, Thermodox®). In that sense, sophisti-
cation of nanoparticles (addition of targeting ligands) would increase
the size of the nanoparticles, leading to a lower entry of these targeted
agents. For instance, MCC-465 and SGT-53 are targeted liposomes
with sizes N100 nm, while BIND-014 and CALAA-01 are polymeric
nanoparticles with sizes of 100 and 75 nm, respectively. Whether in-
creased sophistication of nanomedicines overcomes delivery barriers
posed by their large size will depend not only on the nanoparticle prop-
erties and drug-release kinetics, but also on the tumor type [32]. Elon-
gated nanoparticles with neutral charge should be preferred over
spherical ones since it has been demonstrated that they present an
Non-angiogenic tumor
Dormant
< 1-2 mm Hypoxia
Induction of expression of
Kinin pro-angiogenic proteins:
TNF- VEGF
Kinin-generating cascade
iNOS
MMP
COX PG
Vascular permeability
NO
Normal cell COX: Cyclooxygenase
Cancer cell PG: Prostaglandin
Dividing cell NO: Nitric oxide
Blood vessel with pericyte iNOS: Inducible form of NO synthase
Apoptosing, necrotic cell TNF- : Tumor necrosis factor
MMP: Matrix metalloproteinase
VEGF: Vascular endothelial growth factor
Fig. 4. Schematic representation of the factors involved in the angiogenic switch and consequently in the EPR effect.
Adapted from [77,78].
improved transport through the pores of the tumor vessel wall and in-
terstitial space [32,70]. Additionally, there is evidence that flexible
nanoparticles of low elastic properties might have longer circulation
times [32,71].
5.1. Vascular mediators involved in the EPR effect
Vascular mediators involved in the EPR effect (Fig. 4) are the follow-
ing: (i) Bradykinin (kinin) is a major mediator of inflammation that in-
duces extravasation and accumulation of body fluids in inflammatory
tissues. Kinin is also known to activate endothelial cell-derived nitric
oxide (NO) synthase, leading to an increase in NO (an important medi-
ator of tumor vascular permeability) [11,72]. (ii) NO is produced from L-
arginine by the inducible form of NO synthase (iNOS) in the presence of
oxygen. In cancer, NO is extensively produced from an increased num-
ber of infiltrated leukocytes and tumor cells [11,73]. (iii) Matrix metal-
loproteinases (MMPs) are zinc-dependent neutral endopeptidases that
are highly expressed in tumor cells and play important roles in tumor
invasion, metastasis and angiogenesis. Activation of MMPs induces the
remodeling of the ECM [11,74]. (iv) Prostaglandins (PGs) are lipid
compounds enzymatically derived from arachidonic acid by cyclooxy-
genase (COX). Similarly to bradykinin, PGs are important mediators in
inflammation and can be upregulated by inflammatory cytokines and
kinin. PGs prevent platelet aggregation, leukocyte adhesion and throm-
bosis formation, facilitating extravasation [11,75]. (v) Vascular endo-
thelial growth factor (VEGF) is the angiogenesis factor that is highly
upregulated in most tumors, which plays a crucial role in angiogenesis.
VEGF was shown to exert its action via generation of NO [76]. Thus all
these vascular permeability factors facilitate the supply of nutrients
and oxygen, and thus angiogenesis.
The following section aims to provide an overview of the various
possibilities allowing nanomedicines to better enter the tumors, despite
Inhibitors Pro-activators
1. Endothelial cell activation A
N
G
I
O
2. Basement membrane degradation
G
E
N
I
3. Endothelial cell migration C
S
W
4. Vessel formation I
T
C
H
5. Angiogenic remodeling
Angiogenic
tumor
 F. Danhier / Journal of Controlled Release 244 (2016) 108–121
Fig. 5. Strategies to modulate or to counterattack the abnormal tumor microenvironment have shown promising results in the enhancement of anticancer drug delivery systems, providing
therefore a second wind to nanomedicines in cancer therapy. ACE: angiotensin-converting enzyme; NG: nitroglycerin; NO: nitric oxide; VEGF: vascular endothelial growth factor; TGF:
transforming growth factor; MMP: Matrix metalloproteinase.
an altered and heterogeneous EPR effect. The different approaches are
summarized in the Fig. 5.
5.2. Microenvironment modifications/modulations
5.2.1. Direct permeability enhancement
Inhibitors of angiotensin-converting enzyme (ACE) (Fig. 5) inhibit
conversion of angiotensin I to angiotensin II by carboxypeptidase. ACE
inhibitors (such as enalapril or temocapril) inhibit the degradation of
bradykinin, thus raising the local bradykinin concentration in tumor tis-
sue, leading to activation of endothelial NOS because the level of brady-
kinin is increased. Thus, the EPR effect becomes more apparent, and
such inhibitors enhance delivery of macromolecular drugs or compo-
nents to the tumor [11,76].
Nitroglycerin and other NO-releasing agents (Fig. 5) generate NO se-
lectively in hypoxic tumor tissue compared with normoxic tissues.
Hence, such nitro agents facilitate the EPR effect via local NO generation
in tumors, with drug delivery enhanced 2 to 3-fold with an improved
therapeutic effect [11,76].
One of the main purposes of the vascular normalization (Fig. 5)
strategy is to transform the phenotype of abnormal tumor vasculature
into a phenotype that closely resembles functional normal vasculature
by increasing coverage of pericytes and the basement membrane, a re-
duced IFP, and ultimately decreasing vessel leakiness [52]. VEGF overex-
pression has been observed in most tumors and is well know to
promote angiogenesis. Therefore, VEGF is a therapeutic target for vascu-
lar normalization. VEGF and other angiogenic signaling factors can be
blocked by using a variety of monoclonal antibodies (mAb). The first ap-
proved anti-angiogenic mAb, bevacizumab (Avastin®) and its subse-
quently developed derivative, ranibizumab, have been used in
metastatic colorectal cancer. Bevacizumab inhibits angiogenesis by
interacting with biologically active VEGF and hinders its binding to the
receptors (VEGFR-1/2) [52,79].
117
This concept of normalized vessels suggest that these “normal” ves-
sels could avoid the EPR effect and compromise the delivery of drugs to
tumors. R.K. Jain resolved this paradox by comparing properties of nor-
mal, tumor and normalized vessels. It appeared that normalized vessels
remain permeable to large molecules but only in case of small NP
(b60 nm) [15,80]. Hence, normalization induces a better delivery of
the therapeutic entities by keeping the EPR effect intact and decreasing
the IFP (Fig. 6A). Although mostly described in mouse tumor models
[81–83], several clinical trials have provided evidence of a normalized
vasculature in cancer patients treated with anti-angiogenic agents [1,2].
Transforming growth factor beta (TGF-β) (Fig. 5) can induce the ex-
pression of different key angiogenic factors, including VEGF and MMP-9
expression in macrophages [84]. Therefore, TGF-β receptor antagonists
(including small molecule kinase inhibitors and siRNAs) were the most
frequently used therapeutic agents to inhibit pericyte recruitment and
BM activation [45,85]. These inhibitors increase vessel leakiness and im-
prove the intratumoral penetration of sub-100 nm nanoparticles. TGF-β
inhibitors were also found to lower interstitial hypertension [45].
5.2.2. Indirect permeability enhancement
The compression of blood vessels (Fig. 5) by proliferating tumor cells
in a confined space can reduce the diameter of blood vessels and conse-
quently decrease the blood flow. Therefore the decompression of blood
vessels can be achieved through the induction of apoptosis of specific
anticancer drugs. Successful decompression of tumor blood vessels
was first reported by R.K. Jain and his colleagues, who used Taxanes
(paclitaxel and docetaxel) to induce apoptosis [86]. The term “tumor
priming” was coined by Au's group when they first describe the reduc-
tion of tumor cell density and expansion of the interstitial space, in-
duced by paclitaxel [87,88]. Paclitaxel is particularly interesting since
its action on an increased EPR effect results in (i) enhanced perfusion
resulting from a decreased IFP and transient vascular normalization
(anti-angiogenic effect) and (ii) the decompression of blood vessels
118 F. Danhier / Journal of Controlled Release 244 (2016) 108–121
Fig. 6. Schematic representation of the effects of (A) anti-angiogenic drugs and (B) paclitaxel on the tumor microenvironment and consequently on the EPR effect.
Adapted from [81].
resulting from the induction of apoptosis (cytotoxic effect) (Fig. 6B).
Since it has been demonstrated that paclitaxel induced apoptosis and
reduced the density of intact neoplastic cells in both MCa-IV and
HSTS-26T, the further in vivo determination of blood vessel decompres-
sion has been assessed by measurements of diameter of tumor vessels
in HSTS-26T tumors implanted in transparent dorsal skin fold cham-
bers. At 48 and 96 h after paclitaxel administration (40 mg/kg), the di-
ameter of tumor vessels was significantly increased. This increase in
vascular diameters was associated with reductions in microvascular
pressure and IFP, measured with the wick-in-needle technique [86].
More recently, paclitaxel-loaded polymeric micelles (M-PTX) have
been shown to enhance the blood flow and oxygenation of tumor 24 h
after treatment [89]. These polymeric micelles were shown to be safe
and effective delivery system for paclitaxel, drastically increasing the
maximum tolerated dose of paclitaxel from 13,5 mg/kg to 80 mg/kg,
for the commercial drug Taxol® and M-PTX, respectively [90]. When
injected as pre-treatment 24 h after treatment, M-PTX (80 mg/kg)
were shown to allow a more effective delivery into tumors of three
anti-cancer nanomedicines: polymeric micelles (20 nm), liposomes
(100 nm) and nanoparticles (230 nm). Qualitative intravital microscopy
using dorsal skin-flap window chambers provided clear evidence of en-
hanced permeability of the tumor endothelium in M-PTX pre-treated
tumors. Quantitative studies also supported this hypothesis (Miles
assay, Electron Spin Resonance (ESR) spectroscopy and accumulation
of radiolabeled-paclitaxel-loaded micelles [81]. This strategy has also
been supported by a clinical study of patients with breast cancer, reveal-
ing that paclitaxel decreases IFP and improves tumor oxygenation [91].
Downregulation of VEGF expression was suggested to be partly respon-
sible for this phenomenon. Paclitaxel was found to have broad inhibito-
ry effects on angiogenesis, including the proliferation, motility and cord
formation of endothelial cells, the angiogenic response in vivo, and
VEGF-induced neovascularization [92]. Paclitaxel induces a transient
normalization of the tumor vasculature and, thus, increased perfusion
that accounts for better delivery of chemotherapeutic agents [93,94].
The two requirements of effective paclitaxel tumor priming are its
dose and the time window, sufficient to produce ± 10% apoptosis. As
aforementioned, the toxicity related to the high dose of paclitaxel re-
quired to induce tumor priming can be decreased by using
nanomedicine.
The ECM of solid tumors (Fig. 5) presents a transport barrier that re-
stricts nanoparticle penetration, thereby limiting the efficacy of nano-
sized delivery vehicles for cancer imaging and therapy. The relative ef-
fect of various components of the ECM most likely varies with tumor
type, as ECM differs from tumors of different type and location [95]. Sev-
eral studies have shown that collagenase improves macromolecular
penetration depth [96], while treatment with hyaluronidase yielded
mixed results [97,98]. The enzyme hyaluronidase degrading
hyaluronidan is reported in phase I and II trials to prevent regrowth of
various tumor types and to improve clinical outcome, when given adju-
vant to chemotherapy. The mechanism for the enhanced therapeutic ef-
fect is not well understood, but degradation of the ECM is assumed to
improve the penetration of the drug [15,99]. However, the clinical utility
of enzymatic ECM degradation remains uncertain because (i) elevated
interstitial collagenase levels are associated with a poor patient progno-
sis in a variety of cancers and (ii) ECM degradation may promote cancer
progression and metastasis [88]. The hormone relaxin is also effective in
increasing drug delivery by modifying collagen structure. Relaxin is a
nontoxic hormone secreted in women during pregnancy to induce up-
regulation of MMPs. Relaxin reduces the collagen content by down-reg-
ulating fibroblast activity and up-regulating collagenase synthesis [52,
100]. The proteolysis of the extracellular matrices fuels the process of
angiogenesis and is mediated by a family of proteases known as MMP.
 F. Danhier / Journal of Controlled Release 244 (2016) 108–121
Of the MMP family, MMP-2 and MMP-9 are thought to play a more
prominent role in tumorigenesis. One of the most common strategies
to target MMP consists in the use of tissue inhibitor of MMP (TIMP). An-
other approach is the development of MMP-sensitive drug delivery sys-
tems [51,101,102].
5.3. Multifunctional nanomedicines
5.3.1. Tumor-penetrating peptides
Tumor-homing peptides (Fig. 5), identified by phage library screen-
ing, have recently been recognized for their ability to enhance targeted
drug delivery into tumors [52]. The major example of this strategy is the
cyclic iRGD peptide. The iRGD peptide is constituted from CRGDK/RGPD,
containing a cryptic CendR motif, RGDK/R that possesses CendR-like tis-
sue and cell penetrating activities. Intravenously injected nanomedicine
coupled to iRGD bound to tumor vessels and spread into the extravascu-
lar tumor parenchyma, whereas conventional RGD peptides (CRGDC
and RGD-4C) only delivered the cargo to the blood vessels. In a first
step, the intact peptide binds to the endothelial cell expressing αv
integrins. In a second step, a protease cleaves iRGD and exposes the
cryptic CendR motif RGDK, which can interact with the neuropilin-1 re-
ceptor and thereby increase tumor vascular permeability. Systemic in-
jection with iRGD improved the therapeutic index of drugs of various
compositions including small molecules ((doxorubicin), nanoparticles
(nab-paclitaxel (Abraxane®) and doxorubicin liposomes) and a mono-
clonal antibody (trastuzumab). Indeed, when injected with iRGD,
Abraxane® accumulated 12-fold more in orthotopic BT474 human
breast tumors than Abraxane® given alone, and penetrated far from
the tumor vessels. They also tested doxorubicin in a liposomal formula-
tion (particle diameter = 120 nm) on orthotopic 22Rv1 human prostate
tumor model. The results were similar to those with free doxorubicin
with regard to treatment efficacy, tumor accumulation of the drug
(14-fold), tumor penetration and toxicity. These results indicate that,
in comparison to drug administered alone, the iRGD combination ther-
apy provides equivalent or better antitumor efficacy at a three-fold
lower dose of the drug, with a commensurate reduction in toxicity. Fi-
nally, they tested the iRGD combination regimen in mice bearing tu-
mors derived from BT474 human breast tumor cells, which
overexpress HER2/neu/ErbB2. Co-injection of iRGD resulted in a 14-
fold increase in trastuzumab- positive areas within the tumor. An en-
zyme-linked immunosorbent assay (ELISA) showed that iRGD en-
hanced the accumulation of trastuzumab in the tumors 40-fold.
Combining trastuzumab with iRGD increased the drug potency at differ-
ent trastuzumab dose levels. At a clinical dose (9 mg/kg), the combina-
tion eradicated all tumors in the mice, whereas the equivalent dose of
trastuzumab alone only slowed tumor growth. Thus, co-administration
of iRGD may be a valuable way to enhance the efficacy of anti-cancer
drugs while reducing their side effects, a primary goal of cancer therapy
research [77,103,104].
5.3.2. Stimuli-responsive nanomedicine
To enhance the preferential accumulation of nanomedicine or drug
release in tumors, efforts are increasing to develop stimuli-responsive
nanomedicine (Fig. 5) that use endogenous or exogenous (also called
external) stimuli to overcome the limitation of EPR effect [105]. Endog-
enous stimuli-responsive nanomedicine utilize local tumor microenvi-
ronment factors: (i) Changes in pH: the extracellular pH values in
solid tumors are usually below 7.0, due to the high rate of glycolysis.
(ii) Redox gradients: the redox potential in microenvironments is mul-
tivariate in different tissues. More particularly, tumors present hypoxic
areas and elevated reactive oxygen species (ROS). (iii) Enzyme concen-
tration: enzymes (such as glycosidases, lipase, phospholipases and
MMPs) are overexpressed in tumors. On the contrary, exogenous-re-
sponsive nanomedicine respond to external stimuli: (i) Temperature:
considering the temperature difference between tumor and normal tis-
sues, some nanomedicines have been designed to be triggered
119
specifically in tumors (in that case, the temperature should be consid-
ered as an endogenous stimulus). However, the majority of drug deliv-
ery systems using temperature as stimulus need an external heating
source (Ultrasound, magnetic field, etc.) to improve the drug release.
(ii) Light: photo-sensitive carriers trigger drug release at the desired
target through stimulation by light. (iii) Magnetic field: magnetic
nanomedicines are guided to the target tissue under programmable ex-
posure of an external magnetic field. (iv) Ultrasound: the development
of ultrasonic sensitive nanocarriers for ultrasonography expands ultra-
sound techniques to be unique and effective methods to capture drug
carriers and trigger drug release by tuning the ultrasound frequency,
duty cycles and time of exposure [105,106]. Exogenous-stimuli respon-
sive systems present some disadvantages: they require expensive
equipments, which are not always practical. Additionally, the depth of
tissue penetration is a major issue of these systems. For more details
about these strategies, an excellent review of P. Couvreur and colleagues
is available [107].
6. Conclusion
EPR effect is probably the most important concept in new anticancer
drug delivery systems. Nevertheless, the EPR effect is extremely hetero-
geneous in humans. Thus the basic rationale of the design and develop-
ment of nanomedicines in cancer therapy is failing and it would be
necessary to stop claiming efficacy gains via the EPR effect, while the
tumor targeting cannot be proved in the clinic. First, nanomedicine
should be studied on more clinically relevant tumor models or should
be dedicated only for tumors presenting a strong EPR effect in the clinic,
such as Kaposi sarcoma and head and neck tumors. Alternatively,
nanomedicines can also be used for oncologic applications that do not
require the EPR effect: the decrease of toxicity of chemotherapeutic
agents, the local delivery of anticancer agents and the use of
nanomedicines as a tool for imaging the tumor microenvironment. Fi-
nally, recent evidence supports a considerable barrier role of tumors
resulting in imperfect or inefficient EPR effect. In this context, strategies
to modulate or to counterattack the abnormal tumor microenvironment
have shown promising results in the enhancement of anticancer drug
delivery systems, providing therefore a second wind to nanomedicines
in cancer therapy.
Acknowledgments
This work is supported by the Fonds National de la recherché
Scientifique (F.R.S.-FNRS).
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