Drug Carriers for Vascular Drug Delivery

Abstract

The currently used drug carriers for vascular drug delivery are reviewed. The human vascular system possesses unique physiological features that can be exploited for enhanced and effective targeted drug delivery. Although the thin layer of endothelial cells (EC) lines the interior surface of blood vessels forming an interface between circulating blood in the lumen and the tissue beyond the vessel wall, it can also function as a target for drugs to EC in different vascular areas. ECs overexpress specific cell-surface molecules under various pathological conditions (tumor neovasculature, inflammation, oxidative stress, and thrombosis), which are absent or barely detectable in established normal blood vessels. By coupling unique endothelial surface markers, such as antibodies, specific peptides, and growth factors to a variety of drug carriers, effective active vascular-targeted drug delivery systems can be achieved. This review focuses on the recent advances and strategies for effective targeted vascular drug delivery using a variety of drug-loaded carriers along with new targeting approaches that can be used in the design and optimization of such carriers.

Full text

Critical Review
Drug Carriers for Vascular Drug Delivery
Erez Koren and Vladimir P. Torchilin
Department of Pharmaceutical Sciences, Center for Pharmaceutical Biotechnology and Nanomedicine,
Northeastern University, Boston, MA
Summary
The currently used drug carriers for vascular drug delivery
are reviewed. The human vascular system possesses unique phys-
iological features that can be exploited for enhanced and effective
targeted drug delivery. Although the thin layer of endothelial
cells (EC) lines the interior surface of blood vessels forming an
interface between circulating blood in the lumen and the tissue
beyond the vessel wall, it can also function as a target for drugs
to EC in different vascular areas. ECs overexpress specific cell-
surface molecules under various pathological conditions (tumor
neovasculature, inflammation, oxidative stress, and thrombosis),
which are absent or barely detectable in established normal
blood vessels. By coupling unique endothelial surface markers,
such as antibodies, specific peptides, and growth factors to a
variety of drug carriers, effective active vascular-targeted drug
delivery systems can be achieved. This review focuses on the
recent advances and strategies for effective targeted vascular
drug delivery using a variety of drug-loaded carriers along
with new targeting approaches that can be used in the design and
optimization of such carriers. Ó2011 IUBMB
IUBMB Life,63(8): 586–595, 2011
Keywords vascular targeting; endothelial cells; drug delivery; phar-
maceutical nanocarriers.
INTRODUCTION
In recent years, the vast increase in the number of a variety of
drug carrier systems, which have been explored or already been
approved for use in the clinic, has had a significant impact on the
diagnosis, treatment, and potential cure of many chronic
diseases, including cancer, diabetes mellitus, psoriasis, Parkinson
disease, Alzheimer disease, rheumatoid arthritis, HIV infection,
infectious diseases, cardiovascular disorders, asthma, and drug
addiction. Up to this date, most of the pharmaceuticals used
against certain diseases are based not on the ability of the drug
agent to accumulate selectively in the pathological organ, tissue,
or cell but on the distribution of the pharmaceutical within the
body. Furthermore, the need of a drug to cross various biological
barriers, such as other organs, cells, and intracellular compart-
ments, where it can be inactivated or express undesirable
influence on organs and tissues that are not involved in the patho-
logical process, is also essential. Targeting a drug specific to its
target organ initiates higher tissue selectivity and local concen-
tration, whereas drug levels in nontargeted cells and organs
remain below toxic levels and without side effects to the host.
As suggested by Paul Ehrlich a century ago, a two-compo-
nent ‘‘magic bullet’’ drug targeting platform should be designed
to recognize and bind the target followed by a local therapeutic
effect. Currently, the use of a third component to such a ‘‘magic
bullet,’’ a pharmaceutical carrier, has demonstrated a broad vari-
ety of useful properties. Surface modification of pharmaceutical
carriers, such as liposomes, micelles, nanocapsules, polymeric
nanoparticles, solid lipid particles, and others (1), can be used
to control their biological properties in a desirable fashion and
make them perform various therapeutically or diagnostically
important functions in chorus. Various methods for immobiliz-
ing proteins on the surface of such carriers were previously
described (2), and to date, targeting of drugs using an antibody
or other vector bound to a drug carrier has continued to be
developed in the fields of cancer and tumor diagnostics and
therapy (3–6) as well as in cardiovascular research (7) and other
clinical manifestations. Along with the development of multi-
functional pharmaceutical carrier concepts and designs (8),
mostly for cancer research, the occurrence of cardiovascular
disease and especially atherosclerosis, the challenges of resteno-
sis of arteries after angioplasty, the growth of plaques on stent
implants, and additional aspects of inflammatory processes in
cardiovascular disease have led to the need for a vascular-
targeted therapeutic approach. Along with the ability of the
Address correspondence to: Vladimir P. Torchilin, Department of
Pharmaceutical Sciences, Center for Pharmaceutical Biotechnology and
Nanomedicine, Northeastern University, 312 Mugar Life Sciences
Building, 360 Huntington Avenue, Boston, MA 02115, USA.
Tel: 1617-373-3206. Fax: 1617-373-7509.
E-mail: v.torchilin@neu.edu
Received 11 April 2011; accepted 13 April 2011
ISSN 1521-6543 print/ISSN 1521-6551 online
DOI: 10.1002/iub.496
IUBMB Life,63(8): 586–595, August 2011

vascular endothelium to act both as a barrier and as a potential
target for drug delivery, this approach is based on vasculature
physical–chemical characteristics (e.g., surface charge), its phys-
iology characteristics, and on the unique endothelial determi-
nants exerted from, or present on, the endothelium lumen. To
facilitate targeting, drug carriers comprised of stimuli-sensitive
and/or conjugated with affinity moieties that bind to endothelial
cells (ECs) can be effective for targeting either normal and/or
pathologically altered EC. The use of cells such as neutrophils
as drug carriers, red blood cells as carriers of antineoplastic
drugs (i.e., in the treatment of red blood cell-consuming
tumors), and lymphocytes as carriers of drugs to treat lymphoid
tumors are also potentially promising drug carriers (9, 10).
This review will focus on recent progress, developments, and
novel strategies for targeted delivery therapeutics to and across
the vascular endothelium.
VASCULATURE CHARACTERISTICS IN HEALTH
AND DISEASE
In the vasculature system, the barrier of the blood vessel
wall hinders blood fluid and leukocytes from entering tissues.
The key structural elements that perform this function are the
EC layer that forms the inner lining of the blood vessels,
intima, in combination with the basement membrane that wraps
the basal surface of these cells. The intima regulates a variety
of functions including vascular smooth muscle tone, host-
defense reactions, angiogenesis, and tissue fluid homeostasis.
These functions are controlled by secreted factors from the EC.
For example, nitric oxide (NO), prostacyclin, and additional
factors control the vascular tone and are also able to suppress
platelets aggregation as antithrombotic factors. Urokinase and
tissue-type plasminogen activators are secreted by EC to dis-
solve blood clots through the generation of the fibrin-degrading
protease, plasmin. In thrombosis or other pathological condi-
tions that associated with inflammation process, ECs secrete a
variety of agents including cytokines, reactive oxygen species
(ROS), growth factors, and a variety of chemoattractants. This
secretion along with the exposure of adhesion molecules leads
to leukocyte attraction, adhesion, and transmigration (11). These
secreted/surface-exposed agents can serve as potential targets
for drug delivery, which have been under investigation (12) and
will be discussed later. The endothelial layer, as part of the
blood vessel, forms an effective barrier for the delivery of drugs
to extravascular targets such as tumors, brain, and myocardium.
As endothelial uptake and transcytosis depend on the size of the
transported agent, the barrier function of the endothelium is
inadequate for large therapeutic agents such as proteins or
genetic materials as well as for drug delivery systems (DDSs)
in the range of nanosized particles, liposomes, or other larger
carriers. Physiological and pathological conditions of vascular-
ized solid tumors make its vasculature erratic and highly perme-
able, a phenomena termed enhanced permeation and retention
effect (EPR). It was previously described that long circulating
stealth liposomes and other nanosized carriers were found to
accumulate in vascularized solid tumors (13, 14) through this
effect. EPR targeting is enhanced with increased circulation
time and when nanocarrier size is small enough (less than 200
nm) to pass through the pores of the leaky vessels.
The same mechanism has been found to enhance a carrier’s
delivery into inflammation sites, where the vasculature is also
highly permeable. A disruption in normal vascular endothelial
function can exacerbate the pathology of number of inflamma-
tion-related clinical conditions such as atherosclerosis, ischemia-
reperfusion injury, stroke, hypertension and also thrombosis,
diabetes, acute lung injury, and sepsis. Furthermore, EC dysfunc-
tion may also lead to defects in angiogenesis, which in turn could
either inhibit vascularization during wound repair resulting in
tissue necrosis or pave the way for defective blood vessel
formation. This process can lead further tumor vascularization.
With respect to the tumor vasculature, the microenvironment
of solid tumors differs from normal tissue by its high interstitial
fluid pressure, hypoxia, and low extracellular pH (15). Normal
tissue vasculature is under tight balance between antiangiogenic
and proangiogenic factors. This balance allows the proper
formation and maintenance of the blood vessels network. In the
case of solid tumors, this balance is shifted toward proangio-
genic factors, a process that eventually leads to the formation of
a disorganized vasculature with structural and functional abnor-
malities such as an incomplete endothelial lining, pericyte layer,
and leaky basal membrane (16). Although the vascular network,
which provides substrates and nutrients through the vasculature
system, can be used by tumors to enhance neovasculature
formation, the same network can also be used both to poten-
tially deliver chemotherapeutic agents to cancer cells and to
deliver antiangiogenic agents to the tumor neovasculature.
The vascular network in health and disease also differs in its
morphological and molecular characteristics, related to expres-
sion of specific membrane molecules and the interposition of
cells between abnormal ECs (mosaic vessels in tumor). More-
over, the development of techniques such as in vivo phage
display combined with growing peptide libraries has allowed
for the recognition of different molecules selectively expressed
on the endothelia of different organs. Some of these characteris-
tics will be described in the following paragraphs in related to
their ability to be potential targets for drug delivery or as modu-
lators of the pathological condition when encapsulated and
delivered to the desired targeted site.
STRATEGIES FOR VASCULAR TARGETING
Any carrier, targeted to the vasculature, should recognize
and adhere to the targeted endothelium while withstanding the
hydrodynamic forces. As under various clinical disorders, ECs
overexpress specific cell surface antigens and additional mole-
cules are able to secret a variety of molecules as a response to
altered physiological conditions or chronic disease, these unique
characteristics can be exploited to achieve active vascular
587DRUG CARRIERS FOR VASCULAR DRUG DELIVERY

targeting of drug carrier systems. Currently, immunoglobulin G
(IgG)-type antibodies represent the most popular class of
affinity ligands for targeting because they are well suited for
conjugation with both drugs and drug carriers. When targeting a
carrier to EC, specific and nonspecific interactions might occur
at the cell/particle interface. The specific interactions are con-
trolled by the formation and breakage of molecular bonds
among ligand molecules distributed over the carrier surface and
receptors molecules expressed at the cell membrane. The non-
specific interactions are related to the balance between attractive
and repulsive short-range forces including van der Waals,
electrostatic, and steric forces, which become significant when
the particle is in close proximity to the vessel walls. The fol-
lowing section will describe the main strategies for vascular
delivery, the variety of potentially therapeutic payloads, and the
different ligand/target molecules currently investigated and
developed in this area.
Endothelial Targets
When designing a targeted DDS to the endothelium, a large
variety of molecular determinants on EC surface can be used
for effective therapeutic delivery (shown in Fig. 1). In addition
to the inflammation process and its characteristics, different
clinical conditions can influence EC characterization by diverse
molecular cascade profiles, which may lead to overexpression
of different targets. As this review is focused on the drug
carriers for vascular drug delivery, the various EC targets will
be briefly described.
Endothelial cell adhesion molecules (CAMs) are attractive
candidates for targeted delivery of various carriers to activated
endothelium in vascular pathology. This family of molecules
includes VCAM-1, vascular adhesion molecule-1; ICAM-1,
intracellular adhesion molecule-1; PECAM-1, platelet-endothe-
lial adhesion molecule-1; and P/E selectin. CAMs can both
serve as targets for diagnostic probes and for targeted treatment
of the vascular inflammatory cascade (17). Tumor ECs have a
comparatively high proliferation because of the tumor cell pro-
duction of proangiogenic cytokines, such as vascular endothelial
growth factor (VEGF), basic fibroblast growth factor (bFGF),
transforming growth factor b(TGF-b), and various interleukins
(18). As the proangiogenic state results in the expression of
these surface antigens that are not expressed by normal endothe-
lium, they are good candidate agents for targeted vascular drug
delivery. Other target proteins (as reviewed in refs. 19 and 20)
also include integrins, which are overexpressed in angiogenic
tumor ECs and malignant tumor cells and can be targeted by
RGD ligands; endothelial ectopeptidases (e.g., angiotensin-con-
verting enzyme); caveolar proteins (gp90, gp85, and gp60); and
receptors for growth factors and transport proteins. Furthermore,
by using a phage display technique, additional potential endo-
thelial targets can be isolated (21). The advantage of phage
display method lies in the built-in recognition (and selection) of
sites accessible only to the blood. During the last few years, a
large number of affinity moieties were identified including anti-
bodies, antibody fragments (scFv), natural ligands, hormones,
growth factors, peptides, and others. These agents can easily be
conjugated onto drug carriers and target-specified ECs.
Extracellular Matrix Targeting
In his review (22), Thorpe described various vascular target-
ing agents (VTAs) as cancer therapeutics. One of them is the
extracellular domain of the human coagulation-inducing protein,
tissue factor (TF), which has previously been targeted to tumor
vessels to induce specific tumor vessel thrombosis. The extrac-
ellular domain of TF is not a coagulant while free in the blood
circulation but becomes a powerful and specific coagulant once
targeted by a targeting ligand to tumor vasculature and initiates
the coagulation cascade leading to occlusion of the vessel.
Specific targeting of TF to tumor vessels has been accom-
plished with antibodies and peptides directed against a variety
of tumor vessel markers, including various CAMs, the ED-B
domain of fibronectin, and prostate-specific membrane antigen.
In these studies, the VTAs homed selectively to tumor vessels
and rapidly induced thrombosis. Furthermore, when the immu-
nostimulatory cytokines IL-2 and IL-12 were targeted to tumor
extracellular matrix, the antitumoral effect was significantly
enhanced.
Atherosclerotic Plaque Targeting
Tissue remodeling and angiogenesis are important processes
during atherosclerotic plaque formation as plaque rupture
appears to be strongly related to acute coronary syndromes and
sudden cardiac death. Furthermore, the ability to selectively rec-
ognize atherosclerotic plaques in vivo using suitable binding
molecules is essential. F16, F8, and L19 are novel monoclonal
antibodies, currently in phase I and II clinical trials. These anti-
bodies showed promising results as radioimmunoconjugates and
immunocytokines in patients with cancer and arthritis (23).
These findings open the possibility for various pharmacodeliv-
ery applications using antibody-based delivery of bioactive
agents (e.g., drugs, proinflammatory or anti-inflammatory cyto-
kines, and procoagulant or anticoagulant agents) to atheroscler-
otic plaque for patients with atherosclerosis.
What Drugs Should Be Delivered to the Vasculature?
Targeting of anticancer drugs to the vasculature is considered
to have several advantages over targeting to tumor cells. As
normal ECs are considered ‘‘inactive,’’ side effects on nontar-
geted endothelium should be at minimum. Different tumors are
characterized with similar epithelial phenotypes, and, therefore,
sometimes the same vascular targeting can be used against
different tumors. Also, given that unlike tumor cells, ECs are
genetically stable, it is plausible that these cells will not easily
develop drug resistance. And last, tumor vessels are more acces-
sible to circulating therapeutics injected intravenously than the
cancer cells themselves.
588 KOREN AND TORCHILIN

The use of conventional chemotherapy to kill tumor endothe-
lium has been previously investigated. Conventional chemother-
apy drugs are known to have an antiangiogenic effect in vitro
and in vivo (24). Their administration at low doses at constant
intervals can kill the tumor vasculature and at the same time ex-
hibit low host toxicity. Another treatment approach is to target
key factors required for the formation of new vessels, so-called
antiangiogenic agents, which inhibit angiogenesis. By binding
to vascular endothelial growth factors (VEGFs) these mono-
clonal antibodies inhibit the angiogenesis process. A few
agents have already been clinically approved by the FDA (e.g.,
Bevacizumab and Ranibizumab), whereas others are currently
in clinical trials. Additional approaches take advantage of the
differences between blood vessels in normal tissue and those of
the tumor vasculature by targeting and destroying the existing
tumor vasculature. These agents are called vascular disrupting
agents (VDAs). They destroy tumors by inducing occlusion of
the tumor vasculature, followed by tumor cell starvation due to
the lack of oxygen and nutrients, which in turn leads to tumor
necrosis (22). VDAs are distinguished from antiangiogenic
drugs by targeting existing vessels, which makes them well
suited in the treatment of large bulky tumors. VDAs can be
divided into two types: the ligand-directed VDAs and the small-
molecule VDAs. The ligand-directed VDAs are antibodies,
peptides, or growth factors that specifically bind to structures on
tumor endothelium and thereafter induce occlusion of the tumor
vessel. This group includes fusion proteins, VEGF–gelonin
complex, immunotoxins (monoclonal antibodies to endoglin,
conjugated to the toxin ricin A), antibodies linked to cytokines,
and more. Liposomally encapsulated drugs and gene therapy
approaches can be combined with this VDA approach and will
be discussed later. The small-molecule VDA group exploits the
pathophysiological differences between tumor and normal vas-
culature, that is, increased proliferation, permeability, and a
reduced reliance on the tubulin cytoskeleton to maintain cell
shape. The small-molecule group includes the microtubulin-
destabilizing drugs, Combretastatin A-4 disodium phosphate,
ZD6126, AVE8062, and Oxi4503, and the flavonoid DMXAA.
These agents are in clinical or preclinical development. The
small-molecule treatment approach can be combined with
targeted carriers such as micelles or liposomes and will also be
briefly discussed in the section ‘‘Drug Delivery Carriers for
Vascular Targeting.’’
Antithrombotic agents prevent or reverse different stages of
the coagulation process. Such agents include Abciximab, an
FDA-approved monoclonal antibody against glycoprotein GP
IIb/IIIa integrin receptor, which is used for platelet aggregation
inhibition in patients undergoing percutaneous coronary inter-
vention. Targeted delivery of imaging and antithrombolytic
agents is now among the most promising areas in both drug
Figure 1. Schematics of the vasculature endothelium and selected molecular determinants that play a pivotal role in targeted drug
delivery to the endothelium. In addition to the EPR effect, which is a commonly used approach for vascular and tumor passive
targeting, additional specific endothelium targets are schematically described. Surface-modified drug carriers decorated with specific
antibodies, peptides, or small molecules were shown to accumulate in the endothelium and therefore can serve as potential
drug carriers for a variety of vascular clinical disorders. [Color figure can be viewed in the online issue, which is available at
wileyonlinelibrary.com.]
589DRUG CARRIERS FOR VASCULAR DRUG DELIVERY

delivery and thrombosis. Additional data related to antithrom-
botic agents are discussed in the section ‘‘Antithrombotic
Agents.’’
Delivery of Biotherapeutics
Antithrombotic Agents. Within the vascular network, the blood
is in a delicate balance between freely flowing throughout all
body tissues and coagulation where a blood vessel is injured or
ruptured. Antithrombotic agents that prevent or reverse different
steps of the coagulation process are generally classified into two
groups. Anticoagulants, work mostly upstream to prevent throm-
bogenesis like heparin, warfarin, and its derivates. The second
group of agents is thrombolytics (plasminogen activators) that
work downstream to dissolve a thrombus, slow down, or pre-
vent its formation. This group includes streptokinase, urokinase,
and tissue plasminogen activators. Mostly, serine proteases acti-
vate the endogenous fibrinolytic system, convert plasminogen
into its active form, plasmin, and initiate fibrinolysis (25).
Targeted visualization (imaging) of thrombi and targeted throm-
bolytic therapy are widely investigated. The most popular
construct used for targeting thrombi is a conjugate between a
diagnostic label (e.g., radioactive metal) or thrombolytic
enzyme (urokinase, streptokinase, and tissue plasminogen acti-
vator) and thrombus-specific monoclonal antibody. Haber and
coworkers’ pioneering work described the antibody-directed
urokinase as a specific fibrinolytic agent (26). Still, the use of
proteins and peptides as therapeutic agents needs to take into
consideration the intrinsic properties associated with their nature
as complex macromolecules, which are also foreign to the host
organism. This leads to low stability of a variety of protein
drugs at normal physiological pH values and temperatures, par-
ticularly when these proteins need special conditions that differ
from the normal environment. Among many ways to stabilize
protein/peptide drugs, their encapsulation in a microreservoir
carrier is frequently applied, protecting them from aggressive
influences of the external medium and, in turn, preventing their
action on normal tissues or cells. This type of system includes
liposomes, micelles, polymeric microparticles, and cell ghosts.
Targeted delivery of antithrombotic (thrombolytic) drugs is
expected to increase their stability and efficacy and decrease
side effects, especially in the case of thrombolytic enzymes.
Integrin receptors, present on the surface of platelets, do not
have a ligand-receptive site for attachment of fibrinogen in their
resting state and therefore have already been used for targeted
therapy or targeted delivery of thrombolytic agents. More
details regarding these targeted delivery platforms will be dis-
cussed in the following sections.
Antioxidant Delivery. Oxidative stress plays a pivotal role in
the pathophysiology of cardiovascular disorders, diabetes, ather-
osclerosis, neurodegenerative disorders, asthma, cataract, irrita-
ble bowel syndrome, and in senescence (27). The endothelium
is a major target of injury from ROS that may inflict tissue
damage and support inflammation processes. The abnormally
high levels of ROS are produced by activated neutrophils and
by ECs. Therefore, the endothelium acts as important and essen-
tial target for antioxidants intervention. Oxidative stress progres-
sion can potentially be prevented by antioxidant enzymes such
as superoxide dismutase (SOD) and catalase. SODs are a class
of enzymes that catalyze the dismutation of superoxide (O
2
2
)
into oxygen and hydrogen peroxide. When not neutralized,
superoxide free radicals can react with NOto form the peroxy-
nitrite agent, a strong oxidant that can aggravate vascular oxida-
tive stress, inflammation, vasoconstriction, and thrombosis.
Catalase catalyzes the decomposition of hydrogen peroxide
(H
2
O
2
) to water and oxygen. Large proteins, such as SOD and
catalase enzymes, do not easily penetrate cell membranes,
which limits their efficacy in protecting cells from cellular reac-
tions involving both intracellularly and extracellularly generated
ROS. These enzymes can be chemically modified-stabilized for
delivery to a desired site of action with a carrier to maintain
their function. Diverse modifications of SOD and catalase
including poly(ethylene glycol) (PEG) coupling or liposomal
encapsulation have been designed to solve this delivery problem
and showed protective effect from oxidative stress damage in
animal models (28). Recently, Shuvaev et al. (29) showed that
superoxide dismutase and catalase conjugated with antibodies to
platelet-endothelial cell adhesion molecule-1 (anti-PECAM/SOD
and anti-PECAM/catalase) bind to endothelium, accumulate in
the pulmonary vasculature, and detoxify ROS. These findings
amplify the clinical potential for antioxidant therapy using its
delivery to the endothelium.
Gene Delivery
The delivery of genes into the vascular wall can be of use
for various applications in, such as, the elucidation of athero-
sclerotic pathogenesis and therapeutic gene delivery for the
treatment of cardiovascular diseases. Successful gene transfer to
the vasculature has been achieved headway to clinical trials in a
number of studies (30). Adenoviruses are currently the most
widely used vectors for gene transfer to normal and injured
vascular walls although other vectors, such as retroviruses and
lentiviruses, have also been used for this purpose. Delivery
vectors that are highly potent in terms of gene transduction effi-
ciency should also be safe and easily applied. Nonviral vectors
have recently received focus as gene carriers, but their transduc-
tion efficiency is very low. Efforts have recently been directed
toward improving this aspect using various platforms. Hood and
Cheresh (31) synthesized an integrin antagonist that was
coupled with cationic lipids to facilitate gene delivery to angio-
genic blood vessels in tumor-bearing mice. Similarly, the RGD
sequence-containing peptide was also coupled with phospholipid
for targeted b-galactosidase gene to tumor vasculature (32).
Polyethylenimine was also demonstrated as an effective nonvi-
ral gene delivery when coupled with the recognition sequence
590 KOREN AND TORCHILIN

for integrin, RGD peptide, to deliver luciferase plasmid DNA
into cancer cells (33). As integrin is highly associated with a
variety of human diseases and cellular functions, it serves as a
potential therapeutic target for various drug- and gene-loaded
carriers to specific targets.
DRUG DELIVERY CARRIERS FOR VASCULAR
TARGETING
An effective design of vasculature-targeted drug delivery
carriers for therapeutic and/or diagnostic use is essential to
achieve efficient, defined, and safe therapeutic interventions for
the treatment of the variety of vasculature clinical disorders. The
section ‘‘Strategies for Vascular Targeting’’ had described the
vascular system characteristics and its potential targets, which
can be exploited for effective targeted delivery of pharmaceutical
carriers to the ECs lining the vascular lumen. During the last few
years, vast research, design, and discovery of affinity ligands use-
ful for targeting these unique epithelial cells characteristics were
found. Mainly, these include monoclonal antibodies and their
fragments, peptides, growth factors, and cationic charge-related
entities. By coupling these affinity ligands to drug carriers, an
efficient delivery of a variety of therapeutic and diagnostic agents
to a specific target can be achieved.
The aim of the following section is to screen the currently
available and widely researched drug delivery platforms that
can be specifically modified or conjugated with a large variety
of affinity moieties to target various agents to the vascular ECs.
Figure 2 illustrates the structural design of some currently avail-
able carriers used for vascular delivery.
In this review, we will focus mainly, but not only, on lipid-
based carriers (e.g., liposomes and micelles), which have been
shown to have attractive biological properties such as high
biocompatibility, biodegradability, and contain the ability to
encapsulate and protect drugs or other agents from the sur-
rounding environment and vice versa. These carriers also have
the ability to entrap both hydrophilic and hydrophobic agents.
The most promising approach to increase a carrier’s circulation
time is their coating with PEG, which decreases the carrier’s
opsonization rate and its recognition by liver cells, thus sharply
increasing liposome lifetime in the blood (‘‘stealth’’ liposomes)
(34). In addition to lipid-based carriers, several of the following
vascular delivery carriers including polymeric nanoparticles,
protein conjugates, recombinant fusion constructs, red blood
cells, microbubbles, and viral vectors will be briefly described.
Some of these carriers have already been shown to be poten-
tially/effective regulators of vascular-related clinical pathologies
in vitro and in vivo, including tumor vasculature, vascular
inflammation and its consequences, oxidative stress, and throm-
bosis. Several are currently undergoing clinical studies. Selected
vasculature-targeted pharmaceutical carriers and their specific
characteristics are presented in Table 1.
Figure 2. Drug carriers for vascular drug delivery. [Color figure can be viewed in the online issue, which is available at
wileyonlinelibrary.com.]
591DRUG CARRIERS FOR VASCULAR DRUG DELIVERY

Table 1
Selected vasculature-targeted pharmaceutical carriers and their specific characteristics
Carrier Drug/agent Medical condition Target
Carrier’s
surface ligand Ref.
Liposome Methotrexate, Doxorubicin Malignant lung disease Lungs Specific mAb 7
Liposome Superoxide dismutase Pulmonary oxidative stress Lungs N/A 28
Liposome Streptokinase Acute myocardial infarction Thrombus Nontargeted 35
Liposome CD39 (NTPDase-1) Thrombosis Endothelium CD39 36
Liposome Radioisotopes for imaging Acute myocardial infarction Myocardial infarction Cardiac myosin mAb 37
Liposome Doxorubicin Angiogenesis Tumor neovasculature SP5-52 38
Liposome No drug Thrombotic events
prevention
Activated platelets RGD 39
Liposome Paclitaxel, Cisplatin,
Doxorubicin, Oxaplatin
Cancer solid tumors Tumor endothelial cells Cationic charge 40
Polymersome No drug Cancer ICAM-1 Anti-ICAM-1 antibody 41
Polymeric micelle Combretastatin A4 Cancer Integrins avb3 and avb5 RGD peptides 42
Polymeric nanocarrier No drug Cancer, Inflammation,
cardiovascular disease
Endothelium Anti-ICAM mAb 43
Red blood cells Anti-inflammatory and
anticancer agents, enzymes,
antithrombotics, thrombolytics,
erythropoietin
Inflammation, enzymatic
deficiency, thrombosis
Intravascular (other blood cells,
pathogenic calls, and
macrophages)
mAb, therapeutics,
Poly(ethylene
glycol) (PEG)
9
Peptide conjugate Imaging—diagnosis Cancer avb3 integrins on EC RGD-quantum dot 4
Peptide conjugate SiRNA Cancer Tumor DOTA-SiRNA 4
Cyclic RGD peptide
conjugate
Paclitaxel, Doxorubicin Breast carcinoma
mice model
Tumor RGDyK, RGD4C 12
PEI-peptide conjugate Gene delivery Tumor vasculature aVb3 integrin receptor RGD peptide 33
RGDK lipopeptide Gene delivery Tumor angiogenesis Proangiogenic a5b1 integrin
receptor and mouse tumor
vasculature
RGDK peptide 32
mAb-
111
In complex Imaging—diagnosis Atherosclerosis Atherosclerotic lesions—proliferating
smooth muscle cells
Z2D3 IgM antibody 7
Albumin-FE
3
O
4
Doxorubicin Tumor Lungs External magnetic field 7
Drug conjugate Urokinase, streptokinase,
tissue plasminogen activator
Thrombosis Thrombus Thrombus-specific mAb 26
Radioimmunoconjugates Imaging diagnostics Cancer/arthritis Isoforms of tenascin-C and
fibronectin
F16, F8, L19 mAb 23
Anti-PECAM/enzyme
conjugates
Superoxide dismutase
and catalase
Pulmonary oxidative stress Lungs, pulmonary vasculature Anti-PECAM/SOD and
anti-PECAM/catalase mAb
29
592 KOREN AND TORCHILIN

Liposomes
Liposomes are well-recognized drug delivery carriers (3).
They are artificial monolamellar or multilamellar phospholipid
vesicles of different size and composition that have been recog-
nized as pharmaceutical carriers with great practical potential.
To date, liposomes have been approved by regulatory agencies
to carry a range of chemotherapeutics. These carriers can entrap
practically hydrophilic agents within their internal water com-
partment or small hydrophobic agents in their membrane. Lipo-
somes have also shown the ability to deliver pharmaceuticals
into cells or inside individual cellular compartments. In general,
liposomes are able to passively accumulate into areas with
increased vascular permeability and, therefore, are experimen-
tally suitable to passively target the vascular system under path-
ological conditions such as atherosclerotic lesions, vascular
inflammation, thrombosis, and cancer.
The use of liposomes for targeted delivery of a variety of
agents to the vasculature was previously reviewed by selected
authors (1, 6, 8). One example is the early work by Nguyen et al.
(35), which investigated the thrombolytic efficacy of liposome-
encapsulated streptokinase in a canine model of myocardial in-
farction. The time required to restore vessel patency was reduced
by more than 50% with a liposomal enzyme compared with free
enzyme. In addition to a lower dose needed for the encapsulated
agent, a smaller remnant of thrombi was observed with this for-
mulation. Another example demonstrates the great potential to
reduce both intravascular platelet aggregation and thrombosis
sequelae by entrapping antiplatelet peptides into liposomes (36).
In this work, CD-39-containing liposomes effectively inhibited
platelet aggregation, when platelets were activated by various
agents. Another approach of liposomal delivery to the vasculature
is the delivery of the antioxidant enzymes, SOD and catalase. The
delivery of these agents in an animal model of lung oxidative
stress was shown to modulate the tissue damage (28).
A common approach for liposomal targeting, however,
involves the use of liposomes modified with specific targeting
moieties that have a specific affinity for the affected organ or
tissue. These modifications include the addition of antibodies or
specific targeting peptide moieties on the liposomal surface.
The use of cationic liposomes to enhance their target to the
tumor vasculature will also be briefly discussed.
The use of modified liposomes with specific monoclonal
antibodies against some components characteristic of the cardio-
vascular and tumor vascular system was, and is still, a widely
popular targeting concept and previously reviewed (8, 44).
Although there is a vast literature regarding the characteristics
and potential applications for the use of monoclonal antibodies
to specifically target a variety of carriers, an antibody-modified
carrier has not been approved for human use yet. When design-
ing immunoliposomes, antibodies are conjugated either to the
liposomal surface or to the distal end of the liposomal PEG (2).
We will describe only few examples for the use of immuno-
liposomes to target the vasculature with this commonly used
specific targeting platform.
Khaw et al. described 30 years ago the specificity of local-
ization of myosin-specific antibody fragments in experimental
myocardial infarction (45) and showed the preservation of this
antibody activity after covalent coupling to liposomes (37).
Later on, this group also demonstrated suppressed hypoxic
cardiocyte death by sealing membrane lesions with antimyosin-
liposomes (46).
Another interesting platform for immunoliposomes is their
use as an acoustically reflective carrier that can be targeted for
site-specific acoustic enhancement. Ultrasound parameters to
enhance the delivery of therapeutic-loaded echogenic immunoli-
posomes into the arterial wall for the treatment of atherosclerosis
were investigated in an ex vivo mouse aorta model. By using
anti-ICAM-targeted echogenic liposomes and following the
1-MHz wave ultrasound, greater adherence of the targeted lipo-
somes to vascular endothelium and greater passage across the
vessel wall was shown (47). It was also previously observed that
targeting liposomes to ICAM-1, VCAM-1, fibrin, fibrinogen, and
TF, in addition to application of ultrasound waves, was able to
produce the targeted enhancement in the vessel walls 5 min after
intravenous administration of targeted liposomes.
Several in vivo studies have investigated the tumor vascular
targetability of liposomes decorated with a variety of peptides.
These peptides includes RGD (Arg-Gly-Asp) motifs binding to
avb3 and avb5 integrins (reviewed in ref. 38), NGR motifs that
bind to aminopeptidase-N, CREKA that binds to fibrinogen or
fibrin, GPLPLR that binds to membrane type 1 matrix metallopro-
teinase, peptides binding to unknown receptors such as APRPG,
and the synthetic angiostatic peptide anginex that binds to galec-
tin-1, which is a carbohydrate-binding protein with affinity to
b-galactosidase. Much research is being carried out with peptide-
modified liposomes. Unlike antibodies, peptides have been shown
to have slower clearance from the circulation, and various
researchers have shown the advantage of using these peptides as
targeting moieties on the liposomes surface. RGD peptide, for
example, showed a significant improvement in targeting
liposomes to vascular lesions and to activated platelets (39).
In addition to gene therapy, cationic liposomes were shown
to be a promising carrier system for the delivery of anticancer
agents to tumor ECs. These positively charged carriers take
advantage of the natural affinity of cationic molecules at the
carrier’s surface toward anionic molecules on the targeted cells
surface (e.g., glycoproteins, anionic phospholipids, and proteo-
glycans) in the tumor microvasculature (40).
Other Carriers
Polymersomes contain a similar architecture to that of lipo-
somes, except for the fact they are composed of synthetic poly-
mer amphiphiles, including poly(lactic acid) (PLA)-based
copolymers. Earlier, Hammer and coworkers described their
ability to functionalize polymersomes with an anti-ICAM-1
antibody and characterized the adhesion of the antibody-func-
tionalized polymersomes (41).
593DRUG CARRIERS FOR VASCULAR DRUG DELIVERY

Polymeric micelles are self-assembling monolayers,
formed by amphiphilic block copolymers and demonstrate a
series of attractive properties as drug carriers. These include
high stability both in vitro and in vivo andgoodbiocompat-
ibility. Micelles can also be successfully used for the solubi-
lization of various poorly soluble pharmaceuticals. As drug
carriers, these particles are useful as targeted DDSs.
Recently, a novel targeted polymeric miceller formulation of
Combretastatin A4 (CA4), an antivascular agent, was devel-
oped (42). The lipophilic CA4 agent was entrapped into
RGD-modified polymeric micelles. This micellar formulation
significantly enhanced cell uptake of encapsulated drug by
angiogenictumorECsandalsoresultedinanincreased
antiproliferation effect as a result of the agent’s antivascular
activity.
An additional group of drug carriers includes other poly-
meric nanocarriers. Biodegradable synthetic polymers have
attracted significant attention during the last decade. These
polymeric nanoparticles offer some specific advantages, such as
an increased stability of drugs/proteins and useful controlled
release properties. Nevertheless, concerns are arising from the
use of polymer-based carriers because they include an inherent
structural heterogeneity of polymers, without homogenous size
distribution. When using these nanoparticles, the drug can be
dissolved, entrapped, encapsulated, or attached to their matrices.
The majority of research in this field was done using poly(D,L-
lactide), PLA, poly(D,L-glycolide), poly(lactide-co-glycolide),
and poly(cyanoacrylate) polymers.
Muzykantov and coworkers (43) tested the in vitro and
in vivo parameters of targeting to ECs of anti-intercellular adhe-
sion molecule-1 (ICAM-1)/polymeric nanocarriers consisting of
either prototype polystyrene or biodegradable poly(lactic-co-gly-
colic) acid polymers. Anti-ICAM-1-modified particles bound
specifically to tumor necrosis factor-activated ECs in a dose-
dependent manner and accumulated in the pulmonary vascula-
ture after i.v. injection in mice.
Protein Conjugates
A blood clot is one of the most clinically important
intravascular targets, when met in the lungs or the heart.
Targeted visualization on thrombi and targeted thrombolytic
therapy are of interest both for drug delivery and for identi-
fication of thrombosed areas. The most popular construct
to be used for targeting thrombi is a conjugate between a
diagnostic agent or thrombolytic enzyme (urokinase, strepto-
kinase, and tissue plasminogen activator) and thrombus-
specific monoclonal antibodies. Haber and coworkers (26)
demonstrated that antifibrin antibodies can be conjugated
with urokinase without affecting the specific properties of
the enzyme or antibody. Muzykantov and coworkers showed
that antithrombotic plasminogen activator conjugated with
ICAM-specific IgG demonstrated highly efficient targeting to
the endothelium of mice (48).
Red Blood Cells
The delivery of an agent to the vasculature lumen, or to
phagocytic cells, can be performed using RBCs as the carriers.
Several methods to encapsulate agents into RBCs (including the
hypotonic dialysis) are currently available. By encapsulating
drugs into isolated RBC ex vivo or by coupling agents to the
cell surface, we may be able to use these human cells as car-
riers of a variety of agents. This vascular delivery platform was
previously reviewed and well discussed in ref. 9.
CONCLUSION
Vascular targeting for the delivery of a variety of agents
should improve the safety, effectiveness, and specificity of diag-
nosis and treatment of a plethora of cardiovascular, pulmonary,
genetic, oncological, and other disease conditions. Recent find-
ings described specific surface molecular determinants on ECs
surfaces, typical of healthy and pathological conditions. These
specific receptors, cell adhesion, and additional molecules can
be targets for the delivery of a variety of drug-loaded pharma-
ceutical carriers. By coupling the targeting moieties, such as
antibodies, specific peptides, and growth factors to a drug
carrier’s surface, effective active vascular targeting of DDSs
can be obtained.
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