2

Passive Targeting of Solid

Tumors: Pathophysiological
Principles and Physicochemical
Aspects of Delivery Systems
S. M. Moghimi
CONTENTS
2.1 Introduction ........................................................................................................................... 11
2.2 Barriers to Extravasation....................................................................................................... 12
2.3 Selected Delivery Systems.................................................................................................... 12
2.3.1 Liposomes.................................................................................................................. 12
2.3.2 Polymeric Nanoparticles ........................................................................................... 14
2.3.3 Nanotechnology-Derived Nanoparticles ................................................................... 15
2.3.4 Macromolecular and Related Delivery ..................................................................... 16
2.4 Conclusions ........................................................................................................................... 16
References ...................................................................................................................................... 17
2.1 INTRODUCTION
A solid tumor comprises two major cellular components: the tumor parenchyma and the stroma; the
latter incorporating the vasculature and other supporting cells. As the tumor grows, in order to meet
the metabolic requirements of an expanding population of tumor cells, the pre-existing blood
vessels become subject to intense angiogenic pressure. Several factors produced by tumor cells
and inltrating immune-competent effector cells in the tumor parenchyma are believed to signal the
development of new capillaries from the pre-existing vessels by capillary sprouting and/or dysre-
gulated intussusceptive microvascular growth.
1
Further, in many solid tumors, endothelial cells
destined to create new vessels are recruited not only from nearby vessels, but also to a signicant
extent from precursor cells within the bone marrow (so-called endothelial progenitor cells),
a process referred to as vasculogenesis.
2
Scanning electron microscopy of microvascular corrosion casts has allowed visualization of the
geometry of blood vessel architecture in solid tumors. From these studies, it has become apparent
that tumor blood vessels are highly irregular and show gross architectural changes that differ from
those in normal organs and from newly formed blood vessels, such as those found in wound healing
and in other angiogenic sites.
1,3
For instance, the thickness of a tumor blood vessel wall is poorly
11
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correlated to its diameter. Therefore, despite the large size of some tumor vessels, the tumor blood
ows is chaotic, with high ow rates in some segments and stagnation in others.
1
Also, the blood
ow may temporarily change direction within individual tumor vessels.
Further, the structure and organization of the endothelial cells, pericytes, and vascular basement
membrane of tumor vessels are all abnormal.
1,36
One consistent abnormality of tumor blood vessels
is their high permeability to macromolecules, arising from irregularly shaped and loosely inter-
connected endothelial cells (where the size of fenestrae often ranges from 200 to 2000 nm) and their
less frequent and intimate association with pericytes and the vascular basement membrane.
1,35
Marked variability has been noted in endothelial permeability among different tumors, different
vessels within the same tumor, and during tumor growth, regression, and relapse. The extent of
tumor blood vessel permeability is also controlled by the host microenvironment, and increases with
the histological grade and malignant potential of tumors.
7,8
Given the potency and toxicity of modern pharmacological agents, tissue selectivity is a major
issue. In the delivery of chemotherapeutic agents to solid tumors this is particularly critical,
since the therapeutic window for these agents is often small and the doseresponse curve steep.
Therefore, the idea of exploiting the well-documented vascular abnormalities of tumors, restricting
penetration into normal tissue interstitium while allowing freer access to that of the tumor, becomes
particularly attractive.
2.2 BARRIERS TO EXTRAVASATION
As a consequence of temporal and spatial heterogeneity in tumor blood ow, solid tumors usually
contain well-perfused, rapidly growing regions, and poorly perfused, often necrotic areas.
1,3
As in
normal tissues, diffusive and convective forces govern the movement of molecules into the inter-
stitium of tumors. However, diffusion is believed to play a minor role in the movement of solutes
across the endothelial barrier in comparison with bulk uid ow. Examination of pressure gradients
in experimental tumors has suggested that the movement of macromolecules and particulate
materials out of the tumor blood vessels and into the extra-vascular compartment is remarkably
limited. This has been attributed to a higher-than-expected interstitial pressure, in part due to a lack
of functional lymphatic drainage, coupled with lower intra-vascular pressure.
3
In addition, inter-
stitial pressure tends to be higher at the center of solid tumors, diminishing towards the periphery,
creating a mass ow movement of uid away from the central region of the tumor.
3
For example,
the measured interstitial uid pressure in invasive breast ductal carcinoma was 29G3 mm Hg,
compared with 3.0G0.8 mm Hg in patients with benign tumors and K3.0G0.1 mm Hg in patients
with normal breast parenchyma.
9
Nevertheless, the lower interstitial pressure in the periphery still
permits adequate extravasation of uid and macromolecules.
These pathophysiological characteristics have serious implications for the systemic delivery of
not only low-molecular-weight and macromolecular agents, but also particulate delivery vehicles.
Simply enhancing the plasma half-life of these agents (e.g., long-circulating carriers) will not
necessarily lead to an increase in therapeutic effect.
10
Furthermore, distribution, organization,
and relative levels of collagen, decorin, and hyaluronan also impede the diffusion of extravasated
macromolecules and particulate systems in tumors.
11
Thus, diffusion of macromolecules and
particles will vary with tumor types, anatomical locations, and possibly by factors that inuence
extracellular matrix composition and/or structure.
2.3 SELECTED DELIVERY SYSTEMS
2.3.1 LIPOSOMES
Liposomes are perhaps the best studied vehicles in cancer drug delivery, capable of either
increasing the drug concentration in solid tumors and/or limiting drug exposure to critical target
Nanotechnology for Cancer Therapy 12
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sites such as bone marrow and myocardium.
10,12
For example, Myoceteis a liposomal formulation
of doxorubicin (an inhibitor of topoisomerase II) approximately 190 nm in size that was approved
by the European Agency for the Evaluation of Medicinal Products (EMEA) in 2000 for the treat-
ment of metastatic breast cancer. This formulation provides a limited degree of prolonged
circulation when compared with doxorubicin in the free form. Myocete releases more than half
of its associated doxorubicin within a few hours of administration and 90% within 24 h. Similar to
intravenously injected nanoparticulate systems, liposomes are rapidly intercepted by macrophages
of the reticuloendothelial system.
10
Hepatic deposition of Myocetecould lead to gradual release of
the cytotoxic agent back to the systemic circulation (a macrophage depot system), as well as
induction of Kupffer cell apoptosis.
10
Following apoptosis, restoration of Kupffer cells may take
up to two weeks.
13
A potentially harmful effect is the occurrence of bacteriemia during the period of
Kupffer cell deciency. Although Myocete administration decreases the frequency of cardiotoxi-
city and neutropenia compared with free drug,
14
there is still controversy as to whether liposomal
encapsulation exhibits equivalent efcacy to doxorubicin.
15
Macrophage deposition of intravenously administered liposomes can be markedly minimized
either by bilayer or surface modication.
10,16
Regulatory approved examples include DaunoXome
w
,
a daunorubicin-encapsulated liposome 45 nm in size with a rigid bilayer for HIV-related Kaposis
sarcoma, and Doxil
w
/Caelyx
w
, a poly(ethylene glycol)-grafted rigid vesicle of 100-nm diameter
with encapsulated doxorubicin for HIV-related Kaposis sarcoma and refractory ovarian carcinoma.
As a result of their small size, rigid bilayer, and hydrophilic surface display (as in the case of
Doxil
w
), these formulations exhibit poor surface opsonization, a process that limits vesicle recog-
nition by macrophages in contact with the blood and consequently prolongs their residency time
within the vasculature.
10,16
For instance, Doxil
w
has a biphasic circulation half-life of 84 min and
46 h in humans. In addition, Doxil
w
also has a high drug loading capacity; here doxorubicin is loaded
actively by an ammonium sulfate gradient (as doxorubicin sulfate) yielding liposomes with a high
content of doxorubicin aggregates, which remain highly stable within the vasculature with minimum
drug loss.
17
Therefore, it is not surprising to see that such liposomal formulations exhibit favorable
pharmacokinetics when compared with the free drug. For example, the area under the curve after
a dose of 50 mg/m
2
doxorubicin encapsulated in long-circulating liposomes is approximately
300-fold greater than that of free doxorubicin.
17
In addition, clearance and volume of distribution
are reduced by at least 250- and 60-fold, respectively.
17
However, as a result of their prolonged
circulation times, alternative toxic reactions have been reported with such vehicles. The most
notable dose-limiting toxicity associated with continuous infusion of Doxil
w
is palmarplantar
erythrodysesthesis.
17
Following extravasation into solid tumors, long-circulating liposomes often distribute heteroge-
neously in perivascular clusters that do not move signicantly and poorly interact with cancer cells.
18
Therefore, the efux of drug must followthe process of liposome extravasation at a rate that maintains
free drug levels in the therapeutic range. The rate of drug release from liposomes not only depends
on the composition of the interstitial uid surrounding tumors but also on the drug type and encapsula-
tion procedures. The importance of the latter is highlighted by the observation that extravasated
long-circulating cisplatin-containing liposomes (where cisplatin is loaded passively) lack anti-tumor
activity, whereas cisplatin in free form is capable of inserting cytotoxicity.
19
This is in contrast to the
effective anti-tumor property of the same liposomal lipid composition containing entrapped doxor-
ubicin. It is believed that nonspecic chemical disruption or collapse of the liposomal pHgradient, that
is used to load liposomes actively with doxorubicin, may trigger doxorubicin release.
17
Long-circulating liposomes have the capability to deliver between 3 and 10 times more drug to
solid tumors compared with the administered drug in its free form. If the entrapped drugs are
released from extravasated liposomes, it is very likely that these vesicles inherently overcome
a certain degree of multidrug resistance by the tumor cells. Thus, tumor regression is to be expected
with tumors exhibiting a low resistance factor. With tumors exhibiting higher resistance
levels, due to over-expression of energy-dependent efux pumps such as P-glycoprotein and
Passive Targeting of Solid Tumors 13
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multidrug-resistance-related protein, alternative approaches are necessary. One effective strategy is
to use long-circulating temperature-sensitive liposomes in conjugation with hyperthermia, but this
approach has limited applicability for visceral and widespread malignancies.
20
Others have elabo-
rated on biochemical triggers such as cleaveable poly(ethylene glycol)-phospholipid conjugates to
generate fusion competent vesicles
21
and enzyme-mediated liposome destabilization and pore
formation.
2224
Examples of the latter include long-circulating liposomes with attached protease-
sensitive haemolysin
22
and pro-drug ether liposomes (e.g., vesicles containing phospholipids with
a nonhydrolyzable ether bond in the 1-position), which are susceptible to degradation by secretory
phospholipases.
23,24
For instance, the level of secretory phospholipases (such as the secretory
phospholipase A2) is dramatically elevated within the interstitium of various tumors.
Secretory phospholiapse A2 not only acts as a trigger resulting in the release of encapsulated
cytotoxic drugs from pro-drug ether liposomes, but also generates highly cytotoxic lysolipids
that destabilizes plasma membrane of tumor cells, thereby enhancing their permeability to cyto-
toxic drugs.
24
There are several approaches that exploit active targeting of long-circulating liposomes to
tumor cells, where receptor-mediated internalization is strongly believed to bypass tumor cell
multidrug-efux pumps.
10,16,17,25
These strategies utilize tumor-specic monoclonal antibodies
or their internalizing epitopes, or ligands, such as folic acid, which are attached to the distal end
of the poly(ethylene glycol) chains expressed on the surface of long-circulating liposomes. Never-
theless, with such approaches the delivery part is still passive and relies on liposome extravasation.
2.3.2 POLYMERIC NANOPARTICLES
Abraxaneeis the only example of a regulatory approved (FDA, USA) nanoparticle formulation for
intravenous drug delivery in cancer patients. It is paclitaxel bound to albumin nanoparticles, with a
mean diameter of 130 nm, for use in individuals with metastatic breast cancer who have failed
combination chemotherapy or relapse within 6 months of adjuvant chemotherapy. This formulation
overcomes poor solubility of paclitaxel in the blood and allows patients to receive 50%
more paclitaxel per dose over a 30-min period.
26,27
Unlike Cremophor
w
EL/ethanol or Tween
w
80-solubilized taxanes, acute hypersensitivity reactions, which are secondary to complement
activation, have yet to be reported following Abraxanee infusion. Albumin nanoparticles seem
to interact with gp60 receptors present on tumor blood vessels that transport the nanoparticles into
tumor interstitial spaces by transcytosis, a process that may partly contribute to the effectiveness of
Abraxanee. However, hepatic deposition (Kupffer cell capture) and processing of a signicant
fraction of albumin nanoparticles are most likely to occur. Indeed, after a 30 min infusion
of 260 mg/m
2
doses of Abraxanee, faecal excretion accounted for approximately 20% of the
administered dose (ABRAXIS Oncology, A division of American Pharmaceutical Partners, Inc.,
Schaumburg, IL 60173, USA; 2005), thus supporting a role for hepatic handling and biliary
excretion of albumin nanoparticles (or its components).
Nanoparticles assembled from synthetic polymers have also received much attention in cancer
drug delivery.
28
One interesting example is doxorubicin-loaded poly(alkyl cyanoacrylate) (PACA)
nanoparticles. In vitro studies have indicated that PACA nanoparticles can overcome drug resist-
ance in tumor cells expressing multidrug-resistance-1-type efux pumps.
29
The mechanism of
action is related to adherence of PACA nanoparticles to tumor cell plasma membrane, which
initiates particle degradation and provides a concentration gradient for doxorubicin, and diffusion
of doxorubicin across the plasma membrane following formation of an ion pair between the
positively charged doxorubicin and the negatively charged cyanoacrylic acid (a nanoparticle
degradation product).
29
These observations clearly indicate that drug release and nanoparticle
degradation must occur simultaneously, yielding an appropriate size complex with correct physi-
cochemical properties for diffusion across the plasma membrane. Further developments with
PACA nanoparticles include preparations that contain doxorubicin within the particle core and
Nanotechnology for Cancer Therapy 14
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cyclosphorin, an inhibitor of the P-glycoprotein, at the surface.
30
Similar to liposomes, long-circu-
lating versions of PACA nanoparticles have also been engineered for passive as well as active
targeting to solid tumors.
31
2.3.3 NANOTECHNOLOGY-DERIVED NANOPARTICLES
Nanotechnology is a cross-disciplinary eld, which involves the ability to design and exploit the
unique properties that emerge from man-made materials ranging in size from 1 to greater than
100 nm.
16
Indeed, the physical and chemical properties of materialssuch as porosity, electrical
conductivity, light emission, and magnetismcan signicantly improve or radically change
as their size is scaled down to small clusters of atoms. These advances are beginning to have
a paradigm-shifting impact not least in experimental (e.g., thermal tumor killing) and diagnostic
oncology.
16,32
Examples include superparamagnetic iron oxide nanocrystals, quantum dots (QDs),
inorganic nanoparticles, and composite nanoshells. The surfaces of these entities are amenable to
modication with synthetic polymers (to afford long-circulating properties) and/or to targeting
ligands. However, a key problem with these technologies is toxicity and is discussed elsewhere.
16
Iron oxide nanocrystals are formed from an inner core of hexagonally shaped iron oxide
particles of approximately 5 nm, which express correlated electron behavior; at a high enough
temperature, they are superparamagnetic.
33,34
In addition, dextran or synthetic polymers such as
poly(ethyleneglycol) surround the crystal core. Indeed, it is the combination of the small size and
surface characteristics that allow iron oxide nanocrystals, once injected into the blood stream, to
bypass rapid detection by the bodys defence cells and accumulate in tumor sites by extravasation.
Therefore, they are useful for patient selection, detection of tumor progression, and tracking of the
effectiveness of anti-tumor treatment regimens by magnetic resonance imaging (MRI). These
approaches can be extended for site-specic imaging of tumor vasculature with targeting
ligands. In addition, iron oxide nanocrystals can slowly extravasate from the vasculature into the
interstitial spaces, from which they are transported to lymph nodes by way of lymphatic vessels.
34
Within lymph nodes they are captured by local macrophages, and their intracellular accumulation
shortens the spin relaxation process of nearby protons detectable by MRI. On magnetic resonance
images, those node regions accumulating iron oxide appear dark relative to surrounding tissues.
Indeed, iron oxide nanocrystals can distinguish between normal and tumor-bearing nodes and
reactive and metastatic nodes.
34
QDs are made of semiconductors like silicon and gallium arsenide.
35,36
In these particles there
are discrete electronic energy levels (valance band and conduction band), but the spacing of the
electronic energy levels (band gap) can be precisely controlled through variation in size. When
a photon, with higher energy than the energy of the band gap, hits a QD, an electron is promoted
from valance band into the conduction band, leaving a hole behind. Electrons emit their excess
energy as light when they recombine with holes. Since optical response is due to the excitation
of single electron-hole pairs, the size and shape of QDs can be tailored to uoresce specic colors.
The ability of QDs to tune broad wavelength together with their photostability is of paramount
importance in biological labeling.
35,36
Indeed, QDs stay lit much longer than conventional dyes
used for imaging and tagging purposes and therefore have the potential to improve the resolution of
tumor cells to the single cell level by optical imaging as well as determining heterogeneity among
cancer cells in a solid tumor.
3739
Unlike QDs, where optical response is due to the excitation of
single electron hole pairs, in metallic nanoparticles (e.g., gold) incident light can couple to the
plasmon excitation of the metal. This involves the light-induced motion of all valence electrons.
36
Therefore, the type of plasmon that exists on a surface of a metallic nanoparticle is directly related
to its shape and curvature; so it is possible to make a wide range of light scatterers that can be
detected at different wavelengths. Composite nanoshells consist of a spherical dielectric core (e.g.,
silica) surrounded by a thin metal shell (e.g., gold). Again, by controlling the relative thickness of
the core and shell layers of the composite nanoparticle, the plasmon resonance and the resultant
Passive Targeting of Solid Tumors 15
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optical absorption properties can be tuned from near-UV to the mid-infrared. Of particular interest
is the ability of near-infrared light (7001000 nm) to penetrate through tissue at depths of a few cm
with minimal heat generation and tissue damage. Thus, a recent study demonstrated rapid irre-
versible photothermal ablation of tumor tissue in vivo following administration of near-infrared-
absorbing silicagold nanoshells in combination with an extracorporeal low-power diode laser.
40
2.3.4 MACROMOLECULAR AND RELATED DELIVERY
Polymer-based drug delivery systems also favorably alter the pharmacokinetics and biodistribution
of conjugated drugs and accumulate in tumor interstitium following extravasation.
41
Examples
include SMANCS (a conjugate of the polymer styrene-co-maleic acid/anhydride and neocarzinos-
tatin for treatment of hepatocellular carcinoma), conjugates of various cytotoxic agents (e.g.,
paclitaxel, doxorubicin, platinate, and campthothecin) with polyglutamate and nonbiodegradable
hydroxypropyl methacrylamide.
Other related polymer-based systems in cancer drug delivery include micelles
42,43
and dendri-
mers.
44
For example, Pluronics
w
(copolymers of ethylene oxide and propylene oxide) are capable
of forming micelles, and some members of Pluronic copolymers can overcome multidrug resist-
ance.
42
However, it is becoming clear that Pluronic copolymers can induce complement activation,
even at concentrations below their critical micelle concentration, which may increase the risk
of pseudoallergy in sensitive patients.
45
Dendrimers are highly branched macromolecules with controlled near monodisperse three-
dimensional architecture emanating from a central core.
44
Polymer growth starts from a central
core molecule and growth occurs in an outward direction by a series of polymerization reactions.
Hence, precise control over size can be achieved by the extent polymerization, starting from a few
nanometers. Cavities in the core structure and folding of the branches create cages and channels.
The surface groups of dendrimers are amenable to modication and can be tailored for specic
applications. Therapeutic and diagnostic agents are usually attached to surface groups on dendri-
mers by chemical modication. For example, a recent study has used tagged-dendrimers for in vivo
evaluation of tumor-associated matrix metalloproteinase-7 (matrilysin) activity.
46
Other macromolecular systems for cancer targeting and treatment include various forms of
monoclonal and bispecic monoclonal antibodies against tumor-associated antigens.
47
These can
further be coupled to drugs, toxins, enzymes (as in antibody-directed enzyme pro-drug therapy),
cytokines, radionuclides, etc.
2.4 CONCLUSIONS
The chaotic blood ow in tumor vasculature and the heterogeneous vascular permeability of tumor
blood vessels are among the key barriers controlling passive delivery of macromolecular and
particulate delivery systems into the interstitium of solid tumors. Already compromised by
abnormal hydrostatic pressure gradients, compressive mechanical forces generated by tumor cell
proliferation cause intratumoral vessels to compress and collapse, thus creating further barriers for
passive targeting. Interestingly, tumor-specic cytotoxic therapy, reducing tumor cell number, may
result in more efcient delivery by decompressing these same vessels,
48
but this enhanced perfusion
could provide a route for metastasis. Pathohysiologoical barriers, however, are not fully developed
in micrometastases, and also pose a lesser problem in the diagnostic oncology as well as in drug
delivery to well-perfused and low-pressure regions in larger tumors. Some of the problems may
possibly be overcome by design of long-circulating multifunctional carriers (carriers that contain
appropriate combinations of cytotoxic agents, diagnostic, and barrier-avoiding components) with
biochemical triggering mechanisms.
16
The vascular barrier of the solid tumor is also its Achilles
heel; the nutritionally demanding tumor cells are entirely dependent upon a functional vasculature.
Nanotechnology for Cancer Therapy 16
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For this reason, interest has also been focused on the concept of tumor vasculature as a target rather
than a barrier and is reviewed in this compendium and elsewhere.
4951
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