Advanced Drug Delivery Reviews 64 (2012) 640–665
Contents lists available at SciVerse ScienceDirect
Advanced Drug Delivery Reviews
journal homepage: www.elsevier.com/locate/addr
Modern methods for delivery of drugs across the blood–brain barrier☆
Yan Chen a,⁎, Lihong Liu b, 1
a
b
School of Pharmacy, CHIRI, WABRI, Curtin University, GPO Box U1987, Perth, Western Australia 6845, Australia
Institute of Bioengineering and Nanotechnology, 31 Biopolis Way, Singapore 138669, Singapore
a r t i c l e
i n f o
Article history:
Received 6 August 2011
Accepted 21 November 2011
Available online 28 November 2011
Keywords:
Blood–brain barrier
Drug delivery
Receptor-mediated transport
Cell-mediated transport
Nanoparticles
Liposomes
Pathological conditions
a b s t r a c t
The blood–brain barrier (BBB) is a highly regulated and efficient barrier that provides a sanctuary to the
brain. It is designed to regulate brain homeostasis and to permit selective transport of molecules that are essential for brain function. Unfortunately, drug transport to the brain is hampered by this almost impermeable,
highly selective and well coordinated barrier. With progress in molecular biology, the BBB is better understood, particularly under different pathological conditions. This review will discuss the barrier issue from a
biological and pathological perspective to provide a better insight to the challenges and opportunities associated with the BBB. Modern methods which can take advantage of these opportunities will be reviewed.
Applications of nanotechnology in drug transport, receptor-mediated targeting and transport, and finally
cell-mediated drug transport will also be covered in the review. The challenge of delivering an effective therapy to the brain is formidable; solutions will likely involve concerted multidisciplinary approaches that take
into account BBB biology as well as the unique features associated with the pathological condition to be
treated.
Crown Copyright © 2011 Published by Elsevier B.V. All rights reserved.
Contents
1.
2.
3.
4.
Introduction . . . . . . . . . . . . . . . . . . . . . . . .
Physiology and biology of the blood–brain barrier . . . . . .
Transport routes across the blood–brain barrier . . . . . . .
Biological and pathological properties of BBB for drug transport
4.1.
Physical barrier . . . . . . . . . . . . . . . . . . .
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Abbreviations: a2M, alpha-2 macroglobulin; Aβ, amyloid β; ABC, ATP binding cassette; AD, Alzheimer's disease; AIDS, autoimmunodeficiency syndrome; AJ, adherens junction;
AMT, adsorptive-mediated transport; AMP, adenosine monophosphate; ANG1005, angiopep 2 conjugated with 3 molecules of paclitaxel; Antp, Antennapedia; APP, amyloid beta
precursor protein; ApoE, Apolipoprotein E; ATP, adenosine triphosphate; AUC, area under curve; BBB, blood–brain barrier; BCSFB, blood–cerebrospinal fluid barrier; BSA-NP, bovine
serum albumin conjugated nanoparticles; cAMP, cyclic AMP; CBSA, cationic bovine serum albumin; CBSA-NP, CBSA conjugated PEG-PLA nanoparticles; CED, convection enhanced
diffusion; CHP, hydrophobic cholesterol groups; CMC, critical micelle concentration; CMT, carrier-mediated transport; CNS, central nervous system; CPP, cell penetrating peptide;
CRM, cross reacting material; CSF, cerebrospinal fluid; DT, diphtheria toxin; DTR, diphtheria toxin receptor; EAE, experimental autoimmune encephalomyelitis; EO, ethylene oxide;
EC, endothelial cell; EMF, electromagnetic fields; FBP, fusion sequence-based peptide; g7, similopioid peptide; GMP, guanosine monophosphate; HB-EGF, heparin binding epidermal growth factor; HIRMAb, human insulin receptor monoclonal antibody; HIV, human immunodeficiency virus; HLB, hydrophobic–hydrophilic balance; HSA, human serum albumin; HSP-96, heat shock protein 96; HUVEC, human umbilical vein endothelial cells; ICH, intercerebral haemorrhage; ICV, intracerebroventricular; IgG, immunoglobulin G; IL,
interleukin; INF, interferon; JAM, junction adhesion molecules; LDL, low density lipoprotein; LDLR, low density lipoprotein receptor; Lf, lactoferrin; LMV, large multilamellar
vesicles; LPA, lysophosphatidic acid; LRP, lipoprotein receptor protein; LUV, large unilamellar vesicles; MAP, model amphipathic peptide; MAPK, mitogen activated protein kinase;
MCP, monocyte chemotactic protein; MHC, major histocompatibility complex; MLCK, myosin light chain kinase; MP, mononuclear phagocytes; MRP, multidrug resistant protein; MS, multiple sclerosis; NOS, nitric oxide syntheses; NP, nanoparticles; NVU, neurovascular unit; P97, melanotransferrin; PAI-1, plasminogen activator inhibitor 1;
PHDCA, poly(hexadecylcyanoacrylate); PBCA, poly(butylcyanoacrylate); PEG, polyethylene glycol; PEG-PCL, PEG-polycaprolactone; PEG-G-CSF, PEGylated-recombinant
methionyl human granulocyted colony stimulating factor; PEG-PLA, polyethylene glycol-polylactic acid; P-gp, P-glycoprotein; PKA, protein kinase A; PKC, protein kinase C;
PKG, protein kinase G; PLGA, poly(D,L-lactide-co-glycolide); PO, propylene oxide; PTD, protein transduction domain; PTK, protein tyrosine kinase; Qdots, quantum dots; RAP,
receptor associated protein; RES, reticuloendothelial system; REV, reverse phase evaporation vesicles; RMT, receptor-mediated transport; R123, rhodamine 123; SA, sialic
acid residue; SBP, sequence signal-based peptide; SUV, small unilamellar vesicles; TAT, HIV-1 trans-activating transcriptor; TEM, transmission electron microscopy; TER, transendothelial
electrical resistance; TfR, transferrin receptor; TJ, tight junction; TNF, tumour necrosis factors; tPA, tissue plasminogen activator; VE, vascular endothelial; VEGF, vascular endothelial
growth factor; ZO, zonula occludens.
☆ This review is part of the Advanced Drug Delivery Reviews theme issue on “Delivery of Therapeutics to the Central Nervous System”.
⁎ Corresponding author at: School of Pharmacy, Curtin University, GPO Box U1987, Perth, Western Australia 6845, Australia. Tel.: +61 8 9266 2738; fax. +61 89266 2769.
E-mail address: [email protected] (Y. Chen).
1
L Liu is currently funded as an Australian Postdoctoral Fellow by ARC Discovery Project DP110104599 at Chemical Engineering, Curtin University.
0169-409X/$ – see front matter. Crown Copyright © 2011 Published by Elsevier B.V. All rights reserved.
doi:10.1016/j.addr.2011.11.010
Y. Chen, L. Liu / Advanced Drug Delivery Reviews 64 (2012) 640–665
4.1.1.
Tight junctions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
4.1.2.
Adherens junctions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
4.1.3.
Tight junctions regulation and signalling pathways . . . . . . . . . . . . . . . . . . . . . . . .
4.2.
Impact of pathological conditions on the properties of the blood–brain barrier . . . . . . . . . . . . . . .
4.2.1.
Changes in permeability of the blood–brain barrier and drug transport under pathological conditions
4.2.2.
Changes in the blood–brain barrier transport systems under pathological conditions . . . . . . . .
4.2.3.
Monocyte and macrophage trafficking across the blood–brain barrier . . . . . . . . . . . . . . .
5.
Modern methods of transporting drugs across the blood–brain barrier . . . . . . . . . . . . . . . . . . . . . .
5.1.
Drug transport across the blood–brain barrier via tight junction opening . . . . . . . . . . . . . . . . . .
5.1.1.
Enhanced by biological stimuli . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
5.1.2.
Enhanced by chemical stimuli . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
5.1.3.
Enhanced by physical stimuli . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
5.2.
Transport system-mediated drug delivery across the blood–brain barrier . . . . . . . . . . . . . . . . . .
5.2.1.
Nanocarriers for brain drug delivery . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
5.2.2.
Supramolecular architectures of aggregated amphiphilic molecules . . . . . . . . . . . . . . . .
5.2.3.
PEGylation of nanocarriers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
5.2.4.
Functionalization of nanocarriers for drug transport across the blood–brain barrier . . . . . . . . .
5.3.
Drug transport across the blood–brain barrier via transport vectors . . . . . . . . . . . . . . . . . . . . .
5.4.
Drug transport across the blood–brain barrier via adsorptive-mediated transcytosis . . . . . . . . . . . . .
5.5.
Drug transport across the blood–brain barrier via endogenous receptor-mediated transcytosis . . . . . . . .
5.5.1.
Insulin receptor . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
5.5.2.
Transferrin receptor . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
5.5.3.
Low-density lipoprotein receptor related proteins 1 and 2 (LRP1 and LRP2 receptors) . . . . . . . .
5.5.4.
Diphtheria toxin receptor (DTR) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
5.6.
Drug transport across the blood–brain barrier via inhibition of efflux Pumps . . . . . . . . . . . . . . . .
6.
Cell-mediated drug transport across the blood–brain barrier . . . . . . . . . . . . . . . . . . . . . . . . . . .
7.
Conclusions and remarks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
1. Introduction
The blood–brain barrier (BBB) is a dynamic barrier protecting the
brain against invading organisms and unwanted substances. It is also
the most important barrier impeding drug transport into the brain
via the blood circulation. Despite the rapid development in our understanding of the molecular structure of components of the BBB,
our knowledge in receptor expression at the BBB, advances in medical technology, and breakthroughs in nanotechnology-based approaches, many of the brain or central nervous system (CNS)
associated diseases remain under-treated by effective therapies.
This is not because there is a lack of candidate drugs but due to the
inability of many therapeutic molecules to cross the BBB, the
blood–cerebrospinal fluid barrier (BCSFB), or other specialised CNS
barriers to reach the specific areas of brain [1]. Such a difficulty in delivering therapeutic molecules to the brain or CNS can only be overcome by a concerted effort in understanding the physiology of BBB,
its permeability under different pathological or disease conditions,
and its response to physical and chemical stimuli, as well as the various transport receptors at the BBB and available delivery technologies. As many attempts to transport drugs across the BBB could be
against the natural function of the BBB, effective approaches or
methods should be cautiously assessed with regards to their impact
on the overall protective function of BBB. The purpose of this review
is to explore molecular and biological opportunities at the BBB for
transport of therapeutic molecules across BBB under physiologic
and pathological conditions. In addition, any changes in BBB that
can be utilised, when exposed to physical and chemical stimuli, are
analysed for opportunities to maximise drug delivery. In this review,
we also highlight the modern approaches and present insights into
using ligand-conjugation and nanotechnology to target the BBB via
adsorptive or receptor-mediated transport of drug molecules into
the brain. Particular attention has also been paid to the cellmediated approach which takes advantage of the immunological
surveillance system of the brain, using circulating phagocytic cells
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such as monocytes or macrophages as Trojan horse to deliver drug
molecules into the brain.
2. Physiology and biology of the blood–brain barrier
The brain is well protected and dynamically regulated to provide a
sanctuary for the central nervous system (CNS). There are several gateways to enter brain parenchyma, the most important two are blood circulation and cerebrospinal fluid (CSF) circulation. In the human brain,
there are about 100 billion capillaries in total, providing a combined
length of brain capillary endothelium of approximately 650 km and a
total surface area of approximately 20 m 2 [2]. Any molecules' entry
into the brain via parenteral administration is strictly controlled by
the BBB and the BCSFB. As the surface of BCSFB faces the ventricle that
is filled with CSF, not the blood [3], this, in combination with the high
turnover rate of CSF, leads to continuously flushing the injected drug
(i.e. those injected into the ventricle) back to the blood [4]. The BBB,
therefore, is universally considered as the most important barrier in
preventing molecules from reaching the brain parenchyma via extensive branches of blood capillary networks. The chief anatomical and
functional site of the BBB is the brain endothelium. Physiologically, in
addition to brain capillary endothelial cells, extracellular base membrane, adjoining pericytes, astrocytes, and microglia are all integral
parts of the BBB supporting system. Together with surrounding neurons, these components form a complex and functional “neurovascular
unit” [5] (Fig. 1).
A feature of the BBB is its low and selective permeability to molecules which can be attributed to its unique biological characteristics.
These include:
1) the lack of fenestrationsand with very few pinocytotic vesicles, but
a greater number and volume of mitochondria in endothelial cells
[6–8];
2) the presence of tight junctions (TJ) between adjacent endothelial
cells, formed by an intricate complex of transmembrane proteins
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Y. Chen, L. Liu / Advanced Drug Delivery Reviews 64 (2012) 640–665
Fig. 1. Schematic representation of the blood–brain barrier (BBB) and other components of a neurovascular unit (NVU).
Reproduced with permission from reference [11].
(junctional adhesion molecule-1, occludin, and claudins) with cytoplasmic accessory proteins (zonula occludens-1 and -2, cingulin,
AF-6, and 7H6). They are linked to the actin cytoskeleton [9],
thereby forming the most intimate cell to cell connection. The TJ
are further strengthened and maintained by the interaction or
communication of astrocytes and pericytes with brain endothelia
cells [10];
3) the expression of various transporters including GLUT1 glucose
carrier, amino acid carrier LAT1, transferring receptors, insulin receptors, lipoprotein receptors and ATP family of efflux transporters such as p-glycoprotein (P-gp) and multidrug resistancerelated proteins MRPs [3,11]. Some of these aid the transport
into the brain while others prevent the entry of many molecules;
4) the synergistic inductive functions and upregulating of BBB features
by astrocytes, astrocytic perivascular endfeet, pericytes, perivascular
macrophages and neurons, as suggested by the strong evidence
from cell culture studies [12–14];
5) the lack of lymphatic drainage, and absence of major histocompatibility complex (MHC) antigens in CNS with immune reactivity inducible on temporary demand in order to provide maximum
protection to neuronal function [15]. The BBB has a strict limit
for the passage of immune cells, especially lymphocytes [16] and
its immune barrier is made by the association between BBB endothelia cells and perivascular macrophages and mast cells [17]. Additionally, this immune barrier is reinforced by local microglial
cells [18].
All these characteristics lead to BBB to possess multiple functions
as a physical barrier (TJ), a transport barrier (P-gp), a metabolic or enzymatic barrier (specialised enzyme systems [11,19] and an immunological barrier.
3. Transport routes across the blood–brain barrier
It has been well established that there are several transport routes
by which solute molecules move across the BBB [11,20]. Diffusion of
substances into the brain can be divided into paracellular and transcellular. As illustrated in Fig. 2.a, small water-soluble molecules simply diffuse through the TJ but not to any great extent. Small lipid
soluble substances like alcohol and steroid hormones penetrate transcellularly by dissolving in their lipid plasma membrane (Fig. 2.b).
However, for almost all other substances, including essential materials such as glucose and amino acids, transport proteins (carriers),
specific receptor-mediated or vesicular mechanisms (adsorptive
transcytosis) are required to pass the BBB.
In the case of transport proteins or known as carrier-mediated
transport (Fig. 2.c), there is binding of a solute such as glucose or
amino acids to a protein transporter on one side of the membrane
that triggers a conformational change in the protein, resulting in the
transport of the substance to the other side of the membrane, from
high to low concentration. If compounds need to be moved against
a concentration gradient, ATP may provide the energy to facilitate
the process. Efflux pumps or transporters (Fig. 2.d) are responsible
for extruding drugs from the brain and this mechanism is a major obstacle for the accumulation of a wide range of biologically active molecules in the brain, with the ATP binding cassette (ABC) transporter
P-gp and multidrug resistant protein (MRP) being the principle efflux
mechanism of these agents [21]. Inhibition of P-gp in pre-clinical
studies has enhanced the penetration of paclitaxel into the brain, indicating the feasibility of achieving improved drug delivery to the
brain by suppression of P-gp [22].
Receptor-mediated transcytosis (RMT) (Fig. 2.e) provides a means
for selective uptake of macromolecules. Endothelial cells have receptors for the uptake of many different types of ligands, including
growth factors, enzymes and plasma proteins. For example, insulin
molecules first bind to receptors that collect in specialized areas of
the plasma membrane known as coated pits. When bound to ligand
these pits invaginate into the cytoplasm and then pinch free of the
plasma membrane to form coated vesicles. After acidification of the
endosome, the ligand will dissociate from the receptor and cross the
other side of membrane. RMT has been extensively studied for brain
targeting [23]. Those well-characterised systems include transferring
receptor (TfR), insulin receptor, lipoprotein receptors, scavenger receptors class B type I, diphtheria toxin receptor and glutathione transporter [3].
Adsorptive-mediated transcytosis (AMT), also known as the pinocytosis route (Fig. 2.f), is triggered by an electrostatic interaction between a positively charged substance, usually the charged moiety of
a peptide, and the negatively charged plasma membrane surface
(i.e. heparin sulphate proteoglycans). Adsorptive-mediated transport
has a lower affinity but higher capacity than RMT. The development
of many new drug delivery technologies focuses on AMT [24]. AMT-
Y. Chen, L. Liu / Advanced Drug Delivery Reviews 64 (2012) 640–665
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Fig. 2. Transport routes across the blood–brain barrier. Pathways “a” to “f” are commonly for solute molecules; and the route “g” involves monocytes, macrophages and other immune cells and can be used for any drugs or drugs incorporated liposomes or nanoparticles.
Adapted from reference [11].
based drug delivery typically involves either cationic proteins or cellpenetrating peptide such as Tat-derived peptides and Syn-B vectors.
Last, but not least, cell-mediated transcytosis (Fig. 2.g) is a more
recently identified route of drug transport across the BBB [25], although it is a well established mechanism for some pathogens such
as Cryptococcus neoformans and HIV entry into the brain, known as “
Trojan horse” model [26,27]. This transport route relies on immune
cells such as monocytes or macrophages to cross the intact BBB. Unlike aforementioned transport pathways which normally permit
only solute molecules with specific properties, cell-mediated transcytosis is unique in that it can be used virtually for any type of molecules or materials as well as particulate carrier systems [28].
Due to the unique properties of the TJs, paracellular transport of
hydrophilic drugs is virtually absent and transcellular transport by
passive diffusion is only available to molecules which fulfil certain criteria [4,29,30] such as: 1) molecular weight is less than 500 Da;
2) compounds are unionised; 3) log P value of the drug is close to
2; 4) cumulative number of hydrogen bonds is not more than 10. Unfortunately only a small percentage of drugs fit these criteria [2]. For
other therapeutic molecules, their transport across the BBB will
then have to rely on either the integrity of the BBB or the drug or
drug carrier properties and their interaction with or affinity for receptors expressed at the BBB, as well as other biological or immunological processes occurring at the BBB. In other words, the BBB properties
and related biological processes, and their roles in trafficking various
types of molecules are fundamental to the success of drug transport
across the BBB. This is the reason for the need to gain a thorough understanding of the biological and pathological properties and processes of the BBB.
4. Biological and pathological properties of BBB for drug transport
Recent progress in the study of the molecular biology of the BBB
has led to a greater understanding of the barrier functions under normal physiological and pathological conditions, as well as when the
BBB is subjected to external stimuli. More importantly, this knowledge empowers researchers to develop new strategies for therapeutic
molecules to target and transport across the BBB for treatment of
various CNS associated diseases. This section is focused on the physical barrier and properties of the BBB undergoing pathological changes
which may present potential opportunities for drug transport.
4.1. Physical barrier
The physical barrier of the BBB is a result of the formation of an
elaborated junctional complex by TJ and adherens junctions (AJ) between adjacent endothelial cells [31].
4.1.1. Tight junctions
TJ are located on the apical region of endothelia cells and structurally formed by an intricate complex network made of a series of parallel, interconnected, transmembrane and cytoplasmatic strands of
proteins [32,31]. The high level of integrity of TJ is reflected by the
high electrical resistance of the BBB (1500–2000 Ω cm 2), which depends on a proper extracellular Ca 2 + ion concentration. There are extensive reviews on the TJ elsewhere [31–33]. Here the focus of this
review is placed on some key molecules involved in the formation
and maintenance of TJ and the regulation of the permeability of TJ.
Among the identified molecules associated with TJ, the transmembrane proteins claudins and occludin are most well studied. Claudins
form dimmers and bind homotypically to other claudin molecules in
an adjacent brain capillary endothelia cell [34,35] thus forming the
primary seal of the TJ [31]. On the other hand, occludin is not essential
for the formation of TJ, as indicted in the knockout and knockdown
experiments [9] and its main function appears to be for TJ regulation
and as an additional support structure [10,36]. Of claudins, Claudin-5
has been shown to be involved in size-selective loosening the permeability of BBB in mice [37] with permeability of molecules of size less
than 800 Da affected. However, similar effects were not observed
with barrier function of non-BBB endothelium, such as human umbilical vein endothelial cells (HUVEC) [38]. In another experiment, treatment of claudin-5 by cyclic AMP (cAMP) lead to enhancement of
claudin-5 activity along cell borders, rapid reduction in transendothelial electrical resistance (TER), and loosening of the claudin-5-based
endothelial barrier against mannitol, but not inulin [39]. These suggest that manipulation of claudin-5, or potentially other TJ proteins
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may permit drug transport by altering the “molecular sieve” function
at the BBB but without its total disruption [40].
Junctional adhesion molecules JAM-A, JAM-B, and JAM-C, which
are present in brain endothelial cells, also take part in the formation
and maintenance of the TJ [11] , together with occludin and claudins.
Although functions of JAM at the BBB are still largely unknown, it has
been suggested that their involvement is not limited only to the junctional tightness, they may also associate with leukocyte trafficking,
with implications for immune activity in CNS diseases [41].
Submembranous TJ-associated proteins, also known as peripheral
zonula occludens proteins, such as ZO proteins ( ZO-1, ZO-2 and ZO-3)
are another integral part of TJ with possible functions of forming a scaffold to link TJ to the cytoskeleton [31]. Certainly there is no TJ without
ZO-1 as its molecules are delimiting the interendothelial cleft and connecting transmembranous TJ proteins with the actin cytoskeleton [42].
A recent study in multiple sclerosis (MS) model experimental autoimmune encephalomyelitis (EAE) suggests that the relocalization of ZO-1
from cell boundary to cytosol directly increased permeability of the endothelial monolayer, leading to disruption of the BBB and development
of clinical diseases such as MS [43].
Other cytoplasmic proteins such as cingulin (140 kDa) and 7H6
phosphoprotein (155 kDa) have also been implicated in playing a
role in regulating the permeability of TJ [32]. 7H6 phosphoprotein
has been associated with TJ impermeability to ions and large molecules and evidence suggests that the potential detachment of 7H6
protein from TJ when ATP levels reduce can result in increased paracellular permeability of the BBB [33].
4.1.2. Adherens junctions
Adherens junctions (AJ) are located below the TJ in the basal region of
the lateral plasma membrane. They are composed of transmembrane
glycoproteins (represented by the large family of cadherins) linked to
the cytoskeleton by cytoplasmatic proteins, thus providing additional
tightening structure between the adjacent endothelial cells at the BBB
[31]. In addition to supporting the barrier function, AJ mediate the adhesion of brain endothelial cells to each other, the initiation of cell polarity
and the regulation of paracellular permeability [9]. Cadherins' homotypical interaction with each other takes place when Ca2 + is present.
Cadherin-5, also known as VE-cadherin (vascular endothelial cadherin),
is an important determinant of microvascular integrity. When VEcadherin is over expressed, Ca2 +-dependent cell adhesion, inhibition
of cell proliferation and reduction in cell permeability and migration
are observed [44]. In order for cadherins to work as adhesion molecules,
catenin proteins have to play the role of anchor molecules, linking
cadherin complex to the actin cytoskeleton [33]. One of the catenins,
β-catenin, is a structural protein and also serves as a mediator in regulation of P-gp and other multidrug efflux transporters in brain vasculature [45]. It has also been suggested that its up-regulation is important
for the maintenance of TJ protein assembly and barrier function [46].
4.1.3. Tight junctions regulation and signalling pathways
TJ are highly dynamic structures that undergo changes (breakdown or reassemble) in response to physiological and pathological
conditions, which provides the opportunities for reversible opening
of the membranous barrier, via the use of TJ modulators, to improve
drug delivery across the BBB [47]. Intensive research has been conducted to understand the relationship between TJ function or disruption and the state of TJ protein phosphorylation or dephosphorylation
[48].
Physiologically the opening and closing of the BBB paracellular
pathway is controlled by the dynamic interaction among TJ and AJ
structure elements. It has been suggested that changes in adhesive
properties of TJ and AJ proteins and reorganization of the actin cytoskeleton would result in the formation of intercellular gaps at the
BBB [48]. A correlation between the changes in the adhesive property
of TJ and AJ, and alterations in their phosphorylation status has been
established [31,48]. Phosphorylation, sometimes dephosphorylation
may occur to the TJ proteins as they are phosphoproteins, depending
on the type of stimuli and local microenvironment. For instance, vascular endothelial growth factor (VEGF) induces Ser/Thr phosphorylation, causing redistribution of occludin and ZO-1 in murine brain
endothelial cells [49], whereas, in conditions like calcium depletion
or bacterial infection, TJ proteins (occludin) would undergo dephosphorylation rather than additional phosphorylation during TJ disruption [50]. Similarly, AJ proteins such as VE-cadherin and β-catenin
undergo phosphorylation of Ser/Thr and Tyr residues, which is correlated to opening of the BBB [51]. Such changes in phosphorylation
state of TJ and AJ proteins weaken their interaction, alter transmembrane protein localization and induce their redistribution, eventually
causing the dissociation of junction complex from its cytoskeleton anchor and leading to the increased permeability of the BBB [52,53].
In addition to TJ and AJ proteins, mediators of signalling pathways,
such as protein kinase C (PKC), protein tyrosine kinases (PTKs), nitric
oxide syntheses (NOS), protein kinase A and G (PKA, PKG), myosin
light chain kinase (MLCK) and mitogen activated protein kinase
(MAPK), are also involved in regulation of TJ and TJ-mediated BBB
permeability [31]. They could potentially be exploited for drug delivery. The mechanisms of their function in altering the permeability of
the BBB can be complex and sometimes manifold. For instance, activation of PKC may stimulate phosphorylation of TJ proteins—occludin
or directly stimulate the cytoskeletal contractile machinery by phosphorylating MLCK [54], leading to an increase in the BBB paracellular
permeability. Such activation of PKC may take place in a wide range of
pathological and physiological conditions, such as removal of extracellular Ca 2 +, elevation of intracellular Ca 2 +, and the presence of
Ca 2 + chelators, oxidative stress, inflammatory mediators (e.g. tumour necrosis factors-α: TNF-α), vasogenic agent (e.g. VEGF) and
human immunodeficiency virus 120 kDa ligase (HIV gp 120)
[49,55]. Their action on PKC leads to opening the TJ. Likewise, enhanced PTK's activities have also been associated with increased endothelial permeability under several pathological conditions [58].
Similar to PKC and PTK, activation of MLCK (e.g. by vasoactive mediator histamine and lysophosphatidic acid: LPA) leads to weakening
of the integrity of TJ [56]. Bacteria or viral pathogens, cytokines and
even bile acids are some examples of pathological and physiological
agents that can trigger TJ opening via MLCK [55]. In contrast, pathological conditions such as hypoxia, ischemia or excessive nitric
oxide (NO) release can decrease BBB paracellular barrier function
via activation of PKG, which results in soluble guanylate cyclase activation and elevation of cyclic GMP [57].
Although many signalling pathways participate in TJ opening,
there are exceptions. For example, upon elevation of intracellular
cAMP, PKA can be activated and it stabilises endothelial cytoskeletal
and adhesive structures, strengthens cell-matrix adhesion and inhibits leukocyte adhesion, which eventually results in increased
transendothelial electric resistance (TEER) and decreased TJ permeability [57,58]. There are numerous other signalling pathways and
modulators which can act directly on the TJ components, modifying
permeability of TJ. More comprehensive reviews on this topic can
be found elsewhere [47,31].
To date almost all of the abovementioned studies were carried out
in vitro on brain capillary endothelia cells. Although some of these
processes can be expected to be more complex in vivo, these studies
are important in understanding the development and progression of
many CNS diseases, their pathological conditions with drug transport
opportunities and responses of the BBB to chemical, physical and biological stimuli.
Knowledge of the molecular features of tight junctions regulation
and signalling pathways gained from in vitro studies has been translated into in vivo studies. For instance, in vivo studies have been used
to confirm the location of tight junction-specific protein ZO-1 in the
BBB of rats and humans [59] and to study the underlying mechanism
Y. Chen, L. Liu / Advanced Drug Delivery Reviews 64 (2012) 640–665
of drug action in the brain [60]. In addition, in vivo studies provided
direct insights into the relationship between pathological conditions
and tight junctions regulation or change of signalling pathways
[34,61]. Willis et al. [62] revealed the relationship between transitory
astrocyte loss and the disruption of tight junction complexes in the
rat BBB in an in vivo study using a gliotoxin and three vascular tight
junction markers, providing a better understanding of tight junctions
regulation and the integrity of the BBB.
4.2. Impact of pathological conditions on the properties of the
blood–brain barrier
The properties of the BBB undergo significant changes when the
brain is developing a neurological disorder, in inflammatory conditions or under attack by pathogens. These changes affect the integrity
as well as the functions of the BBB, including transport pathways. In
vitro studies have shown that altered drug delivery under pathological conditions may be mediated through changes in a number of
transport pathways including paracellular transport and transcellular
transport (RMT and adsorptive transcytosis) [3]. Indeed, it has been
anticipated that the impaired BBB may provide a window of opportunity for those drugs which normally are unable to traverse the BBB to
reach the target in the diseased brain. In Table 1, we highlight some of
the examples and the implications of pathological conditions on the
drug delivery opportunities into the brain.
4.2.1. Changes in permeability of the blood–brain barrier and drug
transport under pathological conditions
Analysing the functions of TJ structural components, signalling
pathways and regulation of TJ under different conditions, a large
body of evidence suggests that the permeability of the BBB is strongly
influenced by the stimuli produced by physiological and pathological
conditions such as oxidative stress (e.g. free radical nitric oxide:NO;
peroxide:H2O2), inflammatory mediators (e.g. interleukin-1β, IL-1β;
interferon-γ, INF-γ; TNF-α), lipid mediators (prostaglandin E2 and
F2a), vasogenic agents (e.g. histamine; VEGF), infective agents (e.g.
bacteria and bacteria toxin; viruses and virus components; parasites
645
and fungal pathogens), as well as physiological and immunological
stimuli (e.g. intracellular Ca 2 + and leukocytes, respectively) [47,48].
There is growing evidence that the BBB's integrity is compromised
in brain disorders or diseases such as stroke, Alzheimer's disease
(AD), MS, HIV, Parkinson's, ischemia and brain tumours [11,34,63].
This often leads to disordering, impairment or even breakdown of
the BBB. Often the changes in brain endothelia phenotype, junctional
complex remodelling and a progressive increase in leukocyte infiltration [64] are observed with the breakdown of the BBB. In patients suffering from AD, the level of BBB impairment is closely correlated to
increased rates of neurodegeneration. It was reported by Bowman
et al. [65] that BBB disruption, due to amyloid angiopathy and possible hyperhomocysteinemia, was persistently presented in a subgroup
of patients with AD, which permitted peripheral immunoglobulin G
(IgG) to have greater access to the CNS peripherally. However, the researchers did point out that although the BBB impairment may benefit the delivery of drug treatment, the increase of the BBB impairment
could lead to increased rates of neurodegeneration. In other words, in
the case of AD, restoring BBB integrity maybe more important than
taking advantage of the impaired BBB for drug transport.
In brain disorders such as Parkinson's disease and epileptic seizures, transient “opening” of the BBB occurs due to the transient secretion of inflammatory mediators [66,67]. On the other hand, both
primary and secondary brain tumours can increase BBB permeability,
likely caused by disturbance of TJ complex and/or accumulation of
growth factors such as VEGF and proinflammatory cytokines [68].
Analysing all neuropathological conditions reveals one common factor: inflammation and inflammatory mediators are involved in BBB
disruption. It has been suggested that the process of inflammation
and inflammatory mediators can be targeted or controlled to “open”
or “close” the BBB [48].
It is generally anticipated that BBB interruption that occurs under
various pathological conditions may provide an opportunity for enhancement of drug transport into the brain via the paracellular
route. This, however, has proved to be a more complex issue. Due to
limited in vivo results obtained by different investigators under pathological conditions, the answer is not clear. For instance, Fang and
colleagues recently showed that the enhanced BBB permeability
Table 1
Pathological conditions, their impact on the blood–brain barrier, and drug transport opportunities into the brain.
Pathological condition
Impact on the BBB
Implication for drug traversing the BBB
References
Multiple sclerosis
Disruption of TJ; enhanced leukocyte activity; release of
inflammatory cytokines/chemokines
Potentially it may enhance paracellular transport of drugs.
Alzheimer's disease
BBB disruption and permitted the greater access of
peripheral IgG to the CNS
Overexpression of efflux pumps
BBB disruption
Increase in the diameter of cortical vessels, thinning
of basal lamina, loss of glycoproteins, apoptosis of
endothelial cells and tight junction disruption
Leukocyte invasion, elevated CSF-to-serum albumin
ratio, and BBB impairment
Increased BBB permeability
BBB disruption
Potentially it may enhance paracellular transport of drugs
that have affinity for albumin and IgG into the CNS
Efflux pump inhibitors may improve drug deliver into the brain.
It enhanced therapeutic agent concentration in the brain.
Potentially it may increase drug transport into the brain due
to the leaky barrier.
[291]
[43]
[63]
[65]
Parkinson's disease
HIV
Infectious disease
Inflammation
Stroke
Upregulation of DTR
Trauma
Pain
Brain tumour
Ischemia/seizures
BBB breakdown
Alternation of BBB chemokine receptor due to activated
astrocytes
Decreased TJ proteins and BBB perturbation
Loss of the tight junctions in the tumour vascular system,
enhanced retention effect
Overexpression receptors of folate, insulin and transferrin
Upregulation of DTR
DTR: diphtheria toxin receptor.
[292]
[293]
[294]
It may enhance paracellular transport of drugs and drugs
with affinity for albumin.
It may facilitate paracellular drug transportation.
It enhanced paracellular drug, e.g. Ginkgolide B, passage
into the brain
It may provide disease-induced specific drug targeting of
the BBB and receptor mediated transcytosis.
It enhanced therapeutic agent concentration in brain
It may lead to astrocyte-targeted therapy.
[295,296]
[102]
[30]
[298]
[10]
It may facilitate paracellular drug transportation.
Angiogenic vessels are permeable to nano-sized materials.
[299]
[300]
It enhanced folic acid, insulin and transferrin-attached
nanoparticles across the BBB.
Potentially it may increase disease-induced specific drug
targeting of BBB and receptor mediated transcytosis.
[301,302]
[297]
[69]
[303]
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Y. Chen, L. Liu / Advanced Drug Delivery Reviews 64 (2012) 640–665
associated with stoke or ischemia/reperfusion injury facilitated a significant increase in penetration of Ginkgolide B, a potential neuroprotective agent, through the BBB in a ischemia/reperfusion injury rat model
compared to that in normal rats [69]. However, in a separate study
using MS, AD and, AD related animal models (experimental autoimmune encephalomyelitis, EAE, induced mice for MS, streptozotocininduced mice and TASTPM transgenic mice for AD) showed a significantly enhanced brain penetration rate in EAE mice compared to
naïve mice 10 min after intravenous bolus injection for both paracellular markers—hydrophilic molecule sodium fluorescein (NaF) and atenolol [70]. At 2.5 h post intravenous infusion, concentration of atenolol
was found to be almost identical in EAE and naïve mice and there was
no data reported for other time points post intravenous injection and
for NaF at 2.5 h post intravenous infusion in the study. Data presented
on NaF in naïve and TASTPM mice also showed no differences at 0.5 h
post injection. Researchers of the study concluded that “despite
reported BBB disruption, CNS penetration for small molecule therapeutic agents does not increase in MS- and AD-related animal models”. It is
worth noting that the study, however, did not characterise the level of
BBB disruption in these diseased mice and the comparison data of paracellular markers was collected, in most cases, from a single time point
from a different types of administration (bolus injection versus infusion). It is, therefore, difficult to ascertain the true level of the BBB penetration rate and extent of paracellular molecules given as intravenous
bolus injection in AD and MS conditions.
4.2.2. Changes in the blood–brain barrier transport systems under
pathological conditions
4.2.2.1. P-glycoprotein transporters. P-gp is one of the most important
transport systems as it plays a crucial role in preventing the passage
of drugs and toxins across the BBB as well as facilitating their transport from brain to blood [71]. P-gp expression changes as a result of
pathogenesis, a phenomenon that is associated with a number of
neurological disorders [10]. An inverse correlation was found between P-gp expression and deposition of β-amyloid in AD [72]. Likewise, it was found that there was a decrease in P-gp functional
activity in the brain tissue collected from patients with Parkinson's
disease [73]. However, in some diseases, P-gp is reported to be upregulated. For instance, the overexpression of P-gp was described in epilepsy and P-gp was found with both endothelia cells and perivascular
astrocytes, which was suspected to have contributed to the multiple
drug resistance of the treatment [74]. A similar upgrading of P-gp
was also found at the BBB after focal cerebral ischemia [75]. Sometimes active efflux transport by P-gp can be the major obstacle for delivering a new drug therapy, therefore, to facilitate the treatment,
either P-gp inhibitor should be considered or the drug has to be
chemically modified or formulated in such a way that it will not be
the substrate of P-gp.
One of the success stories of an approach with P-gp inhibitors can
be highlighted by the research using Pluronic block copolymers as an
inhibitor of drug efflux transporters expressed at BBB, for drug delivery into the brain. When co-administrated with drugs, Pluronic ® P85
increased the transport of the drugs that are substrates of these efflux
transporters into the brain [76]. It has been pointed out that the P-gp
inhibitor approach is better suited to improve the delivery of the
treatment to acute diseases such as brain tumour when the aim of
the delivery is to maximise the drug concentration for a relatively
short duration. The prolonged inhibition of P-gp, in the case of chronic administration, may lead to the interference of physiological homeostatic regulation provided by P-gp [77]. Furthermore, blockade
of P-gp in the BBB by application of P-gp inhibitor can lead to a significant increase of brain concentrations of various drugs. This is due to
P-gp's function as efflux transporters in the BBB, which if blocked may
cause dramatic toxicity of drugs in brain so that doses of drugs that
are normally well tolerated may become neurotoxic [78]. Given the
protective role of P-gp in various cells and organs, the prolonged
application of P-gp inhibitors, especially if they are poorly selective,
also poses the risk of systemic toxicity caused by the reduction in
elimination of drugs [79]. Thus the strategy of using P-gp inhibitors
to enhance drug transport into the CNS should be applied with caution and restricted to transient application.
4.2.2.2. Adsorptive-mediated transcytosis (AMT). Changes in AMT
(pinocytosis) and RMT in diseased states were also reported in cell
studies and animal models. These changes will not only affect the
process itself but also affect the drug delivery strategy. An in vitro
study with brain endothelia cells showed an increase in AMT after
treatment with proinflammatory cytokines TNF-α and interleukin-6
(IL-6), which are important cytokines presented in several neurological diseases including MS and AIDS [80]. Increased endocytosis and
immunological responses were reported in rats after head injury
[81]. In another study, Fillebeen and colleagues demonstrated a
marked increase of transcytosis of lactoferrin (Lf), an iron-binding
protein against infection and severe inflammation, at the BBB in
vitro in the presence of TNF-α [82]. It was concluded that such
enhancement of transcytosis did not involve the up-regulation of
the Lf receptor but rather an increase in the rate of transcytosis transport. Furthermore, enhanced endocytotic activity has also been
reported in a rat ischemia/stroke model in which transient focal ischemia was induced using the filament occlusion of middle cerebral
arteries [83]. Although this enhanced endocytosis or transcytosis
mechanism is yet to be confirmed in human with CNS disorders, it
is hypothesized that the same may be expected given that the same
inflammatory mediators or responses are associated with the CNS
diseases in human. Thus, this enhanced endocytosis activity associated
with diseased brain supports the rationale for designing drug delivery
systems with cationic proteins or peptides (e.g. cell-penetrating
peptides) as a means to maximise the drug transport into the diseased
brain.
4.2.2.3. Receptor-mediated transcytosis (RMT). RMT is an important
transport pathway for endogenous peptides such as insulin, insulinlike growth factor and transferrin [84]. It is highly specific and it uptakes macromolecules presented on the luminal side of the brain endothelia cells and delivers them to the brain with the receptor
recycled back to the luminal membrane [29]. Although more and
more receptors have continuously been discovered, differential expression, distribution and regulation of various receptors at BBB in
the diseased brain is not fully understood or characterised. This is
an issue that requires urgent attention as it has a profound influence
on the effectiveness of the targeting strategy of many emerging drug
delivery systems, such as the molecular Trojan horse approach. Currently there is only limited knowledge of how different CNS disorders
impact on the expression of receptors and their regulation despite
this being an important factor which must be considered in the
early stage of designing a BBB targeting approach for a particular
CNS disorder. Here the review will focus on some of the most commonly known receptors involved in RMT.
1. TfR. TfR mediates the transcytosis of transferrin-bound iron
through the brain capillary endothelial cell in humans [85]. It is
the most well characterised receptor. As there is often an association between iron accumulation and cellular damage, iron has
long been considered to play an important role in exacerbating
the brain tissue degradation process in many neurodegenerative
disorders. There is an increased expression of TfR in brain ischemia
by brain capillary endothelia cells [86]. However, a decreased expression of TfR was observed in the hippocampus of humans
with AD compared to that of normal human [87], which supports
an early finding by Kalaria et al. [88] that TfR density was decreased in some cortical areas including the hippocampus in AD
Y. Chen, L. Liu / Advanced Drug Delivery Reviews 64 (2012) 640–665
but relatively unchanged in cerebral microvessels. In contrast, evidence indicates that there is a marked increase in the brain TfR
level during brain injury after intracerebral haemorrhage (ICH)
[89]. Recht et al. reported that an immunohistochemistry study
carried out on normal human brain-tissue and brain tumour biopsy specimens from patients revealed TfR primarily in brain endothelia cells with a differential immunostaining for TfR between
normal and neoplastic tissue, suggesting TfR could be exploited
as a target for delivering drug across the BBB for brain tumour
treatment [90].
2. Insulin receptor: This is the receptor that plays a crucial role in diabetes and obesity. Its density and sensitivity could be altered during the progression of diseases such as AD. Frolich et al. [91]
reported in 1998 that brain insulin receptor density was increased
in patients with sporadic AD compared to the age-matched control
but decreased compared to middle-aged control. It has also been
reported that AD pathological mediators such as amyloid-β (Aβ)
peptide can also compete for insulin binding to the insulin receptor, interfering with insulin metabolism and leading to impaired
glucose utilisation in the AD brain [92]. This evidence strongly suggests that targeting the insulin receptor for the purpose of treating
a brain disease may result in disturbance of insulin metabolism
and may affect insulin utilisation in the brain, therefore, it must
be used with caution.
3. Lipoprotein receptors: Low density lipoprotein receptor (LDLR), lipoprotein receptor-related protein (LRP) 1 & 2 are all related and
expressed on the BBB. They are multifunctional, multi-ligand scavenger and signalling receptors [93]. LRP has been reported to serve
as a receptor for amyloid beta precursor protein (APP), apolipoprotein E (ApoE) and alpha-2-macroglobulin (α2M), all of which have
been genetically linked to AD [94]. LRP is therefore, thought to contribute to the pathobiology of AD. The levels of LRP that are downregulated substantially with age [95] are, a major risk factor for
nonfamilial AD. Such down regulation of expression of LRP decreased the LRP-mediated clearance of Abeta–anti-Abeta complexes, contributing to AD development [96]. On the other hand,
increased expression of LDLR was observed in the hippocampus
of stroke-prone spontaneously hypertensive rats associated with
BBB impairment [97]. It has been suggested that because LRP inhibits the inflammatory process, the expression of LRP may be
affected by most brain diseases which are associated with inflammatory process such as AD, Parkinson's disease, MS and encephalitis [3]. This may explain some of the success seen with LRPtargeted systems such as melanotransferrin/P97 and RAP transport
systems
4. Diphtheria toxin receptor (DTR): DTR also known as the precursor
of heparin-binding epidermal growth factor ( HB-EGF). It is a well
characterised internalizing transport receptor on the BBB, neuro,
and glial cells [98]. DTR involves receptor-mediated endocytosis
with some unique properties. It has no endogenous ligands, thus,
neither competes with nor interferes with endogenous ligands or
transport of essential nutrients into the brain [99]. Since upregulation of DTR expression has been reported in many inflammatory conditions induced by brain diseases [100–102], it has been
proposed as a useful receptor for site-specific disease targeting
[99].
From the above review, it can be concluded that designing or
selecting an appropriate drug delivery system for transporting drugs
across the BBB, should take into consideration the impact of the diseased brain on the transporter or receptor systems. If a receptor is
amplified or up-regulated under pathological conditions, it will aid
drug transport into the brain via RMT. If the receptor is responsible
for transporting nutrients or metabolites, it will not be a good candidate target for drug delivery across the BBB. One also needs to be
aware that receptor-mediated endocytosis has high affinity but may
647
not necessary guarantee efficient transcytosis. Yu and colleagues
demonstrated recently that low-affinity anti-TfR antibody variants
showed a higher level of transcytosis and permitted more to reach
the mouse brain to produce therapeutically relevant concentration
compared to high affinity anti-TfR antibody [103]. Their design of
bispecific antibody proved to be effective in delivering the antibody
against enzyme beta-secretase into the brain for treating AD.
4.2.3. Monocyte and macrophage trafficking across the blood–brain
barrier
Perivascular macrophages, which reside on the parenchymal side
of endothelia cells, close to astrocyte endfeet, originally come from
circulating phagocytes such as monocytes and have shown a remarkable capability to cross an intact BBB with 80% turnover in 3 months
[104]. Such frequent migration of perivascular macrophages across
the BBB plays an important role in the innate and adaptive immune
response for protecting the CNS from pathogens. Interestingly monocytes/macrophages have been suggested as being utilised by pathogens as a vehicle to enter the CNS [26]. This transport route is also
named as “Trojan Horse” mechanism and accepted as one of the
means for pathogens invasion of the CNS [27]. In a recent rodent infection study Charlier et al. demonstrated that bone marrowderived monocytes infected with C. neoformans produced a 3.9 fold
increase in CNS infection compared to the treatment with free yeast
[105], highlighting the prominent role that monocyte trafficking
plays in CNS infection by C. neoformans. In addition, there is compelling evidence to suggest that transmigration of infected monocytes
through the BBB is one of the major mechanisms for HIV infection
of the brain [106].
When the brain is overtaken by neuroinflammation caused by
disorders such as stroke and MS there is evidence showing that the
inflammation process can lead to a breakdown of the BBB and increased trafficking or migration of some immune cells, such as leukocytes including monocytes and macrophages [107,108]. It has been
suggested that monocyte migration through the disrupted cerebral
endothelial cell (EC) junctions plays an essential role in the formation
of MS demyelinating lesions [108]. Furthermore, monocyte adhesion
and subsequent migration in MS was found to be predominantly regulated by VCAM-1, an adhesion molecule expressed on brain capillaries [109].
In the case of brain tumours, histological analysis of gliomas has
shown a high level of macrophage and microglia infiltration
[110,111]. Using erythrocyte bound IgG and tumour homogenate,
Morantz et al. [112,113] quantified the macrophage content in 11
glioblastomas and found values ranging from 8 to 78% with a mean
of 45%. In another study, endogenous macrophages were detected
within the tumour or tumour periphery but not in the nontumoural hemisphere 2 weeks after implantation of a tumour in a
rat brain [114]. These findings suggest that macrophage and microglia
accumulation in tumours such as glioma occurs naturally and could
be a part of the immune defence mechanism in response to pathological conditions. It was suspected that a chemokine, monocyte chemotactic protein (MCP-1), which was found in glioma cells might have
promoted monocyte and macrophage infiltration of glioma [115].
One can speculate that this naturally preferential brain tumour targeting property of monocytes and macrophages provides a promising
opportunity for delivery of anticancer drug across the BBB via monocyte or macrophage cell-mediated transcytosis, and directly into the
tumours.
Furthermore, Djukic et al. demonstrated that in the case of bacterial meningitis, there is an increased transmigration of monocytes
across the BBB and their differentiation to microglia [116]. By tracking
fluorescently labelled monocytes in the diseased brain, the study also
showed that these newly recruited monocytes became an integral
part of the pool of parenchymal microglia and contributed to the
clearance of damaged tissue.
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Y. Chen, L. Liu / Advanced Drug Delivery Reviews 64 (2012) 640–665
These studies provide evidence to support the rationale that the increased trafficking of circulating cells such as monocytes/macrophages
across the BBB in the disease brain should be fully utilised and exploited
as a potential cell-mediated transport mechanism and their role in maximizing the effectiveness of a drug delivery system for the treatment of
CNS diseases warrants further investigation and exploration. Recently
there are increasing efforts directed towards this area and this will be
one of the focuses in this review on modern approaches of drug delivery
across the BBB.
5. Modern methods of transporting drugs across the
blood–brain barrier
As previously stated, the various transport mechanisms across the
BBB can be summarised as per Fig. 2. The paracellular pathway, transcellular pathway, transport proteins, efflux pumps, RMT and adsorptive transcytosis described in Fig. 2 have long been accepted as
potential transport routes for drug entering the brain [20]. The cellmediated transcytosis is a relatively new approach for transporting
therapeutical materials that emerged in last decade [25,117]. The
rapid progress in molecular biology has propelled the development
of novel drug delivery systems that take advantage of our better understanding of the BBB, the brain and various brain disorders. There
is an increase in multi-discipline approaches combing biology, nanotechnology and even biophysics to achieve the common goal. With
this in mind, the following section will focus on the latest developments in TJ opening, receptor-mediated, adsorptive-mediated, efflux
pump inhibition and cell-mediated transportation approaches.
5.1. Drug transport across the blood–brain barrier via tight junction
opening
In the past two decades, there has been gradually emerging
knowledge and understanding of molecules involved in TJ and AJ,
and parallel discovery of modulators which can be used for opening
the BBB, ranging from chemical and biological substances to physical
stimuli such as high frequency focused ultrasound and electromagnetic fields (EMF) [47,118,119]. Table 2 presents examples of a wide
range of these modulators and their impact on the BBB and drugs or
tracers passage through the BBB.
The rationale for modulating TJ opening to enhance the paracellular
approach is fourfold: 1) the TJ opening or BBB leakage is a phenomena
associated with many brain diseases (Table 1) and stimuli, and many
modulators have already been characterised; 2) enhanced paracellular
transport will increase the delivery of small water soluble molecules
into the brain; 3) modulated TJ opening may also improve the BBB passage of macromolecules and drug delivery systems including liposomes,
nanoparticles, micelles, polymer conjugates, and their distribution in
the brain; 4) using physical stimuli such as ultrasound and EMF will
temporarily provide local BBB disruption, therefore, concentrated drug
can be delivered locally.
5.1.1. Enhanced by biological stimuli
Several modulators with capacity to temporarily open TJ to enhance the transport of drugs and traces have been reported and
have been mostly studied on in vitro cell models (for review see
[57]). It is interesting to note that a 45 kDa biological molecule zonula
occludens toxin (Zot), an active TJ modulator at the BBB, can induce a
reversible, concentration-dependent TJ opening (measured by TEER
decrease), which increases the paracellular transport of sucrose and
inulin (permeability markers) without detectable short-term toxicity
in cultured bovine brain capillary endothelia cells [120]. In addition, it
also permits an enhanced transport of the therapeutic agents doxorubicin and paclitaxel that are substrates of P-gp and otherwise would
have very low transportation across the BBB [120].
Other groups of biological compounds which can act on TJ are vasoactive compounds and inflammatory stimuli such as histamine,
bradykinin and VEGF. They are products or mediators of the inflammation process and can increase BBB permeability [54,121]. In the
case of brain tumours such as gliomas, microvascular permeability
in tumour tissue is more sensitive to the effects of these biological
compounds than the normal brain endothelia cells [122]. Therefore,
these stimuli, when used in combination with imaging materials,
gene or anticancer drugs, can potentially boost the preferential delivery of these materials to the brain tumour to achieve tumour diagnosis, tumour gene therapy or chemotherapeutic treatment. This
hypothesis is supported by the experimental results obtained on a
rat glioma model which showed Intracarotid histamine infusion selectively increases permeability of Evans blue albumin and enhanced
the transport of α-aminoisobutyric acid in brain tumours without affecting BBB permeability in the normal brain tissues [123,124]. It is
suggested that the signalling pathway for histamine's effect involves
H2 receptors, NO and cyclic GMP production [124–126].
Table 2
Effect of selected stimuli/agents on the blood–brain barrier (BBB) and drug transport.
Stimuli/agents
Chemical
Cyclodextrin
Poloxamers (Pluronic® block
copolymers)
Cell penetrating Peptides
Peptide
Biological
Virus
HIV-1clade-sepcific Tat protein B
Macrophage
Cereport (RMP-7)
Physical
Ultrasound
Microwave
Electromagnetic field
BCEC: brain capillary endothelial cells.
Effect on the BBB
Effect on drug/marker transport across the BBB
References
Extracting cholesterol from BCEC membrane leading
to opening of tight junctions
Inhibiting P-gp and MRP efflux transporters at the
concentration below critical micelle concentration
inconsistent
High percentage of DOX across in vitro brain endothelia
cell monolayer
Its co-administration with digoxin enhanced the drug
delivery into the brain.
Cannot cross intact BBB; transport drug via adsorptivemediated transcytosis
Delivered siRNA to the neuronal cells
[155]
Binding to neuron cell acetylcholine receptor
Upregulation of chemokines to open tight junctions
Disrupt BBB integrity
Trojan horses effect
Open tight junctions
Transcytosis ; transendothelial openings; partial
opening of tight junctions
Increasing BBB permeability via thermal effects
Opening tight junctions via protein kinase C signalling
and tight junctions protein translocation
Permitted passage of Glutamic acid decarboxylase (GAD)
gene with adeno-associated virus (AAV) safely improved
condition in AD patients
May increase paracellular transport
Facilitated brain delivery of didanosine and indinavir
Enhanced the CNS delivery of carboplatin, loperamide
and cyclosporin-A
[276]
[304]
[305]
[139,145]
[306]
[307,308]
[130,135,128,127,134]
Enhanced delivery of antibody, chemotherapy and gene
[309–311,118]
Enhanced horseradish peroxidase transport in a rat model
Increased BBB permeability of saquinavir in an in vitro
BBB model
[165]
[177,178]
Y. Chen, L. Liu / Advanced Drug Delivery Reviews 64 (2012) 640–665
Cereport (also known as RMP-7 or Lobradimil), a synthetic peptide analogue of bradykinin, has been extensively studied for facilitating drug transport via the paracellular route [127–130]. Cereport
increases the permeability of the BBB by transiently disrupting the
TJ [131]. It shows specific time, dose and size dependent actions on
human brain microvascular endothelial cells and its effect can be
modulated through changes in cAMP and cGMP second messenger
systems [132]. Cereport is also unique in that it is effective when it
is given via either intracarotid or i.v. administration, although the latter does require a higher dose [128,133]. Studies have shown that
Cereport is capable of enhancing the BBB transport of a number of
drugs including carboplatin, loperamide, and acyclovir in different
types of diseased animal models [127,128,134]. Because Cereport
has no toxicity by itself, can selectively increase drug uptake in the
brain tumour and showed less effect in non-permeable normal
brain, it was once held as one of the most effective BBB modulators
despite its inability to inhibit the P-gp efflux pump, therefore no effect on BBB transport of drugs which are P-gp substrates such as paclitaxel [135]. Nevertheless, at a high dose, Cereport did show an
enhanced survival rate in rat glioma model when combined with carboplatin [135].
When attached to the surface of a liposome, Cereport is even more
effective in facilitating Evans blue transport into the brain compared
to free Cereport with liposome [136]. The latter was ineffective due
to the different arrival times of free Cereport and Evans blue incorporated liposomes at the BBB, which results in inefficient opening of BBB
for Evans blue incorporated liposomes. This study also compared the
bioactivity of Cereport-attached liposomes with free Cereport in vitro
on mouse brain microendothelial vessel cells and showed that the
former was a little stronger than that of free Cereport. This study
also demonstrated that Cereport attached liposomes can be potentially used for transporting different types of drugs, including P-gp substrates via transient TJ opening.
Despite positive results obtained with Cereport in facilitating carboplatin in animal models and phase I clinical trial, the outcomes of a
phase II clinic trial were inconsistent. The combination showed a significant activity in recurrent malignant glioma patients following radiotherapy [137], whereas, it was found to be inactive in childhood
high-grade gliomas and brainstem gliomas [138]. This highlights the
challenge of translating experimental research into a clinical therapy.
Nevertheless, Cereport still warrants further research in enhancing
drug transport across the BBB.
Virus is also one type of biological material which can act as stimuli
and open the TJ via upregulation of chemokines as a precursor for infiltration of inflammatory cells into the CNS [139]. Immunohistochemical
analysis of CNS tissue from HIV-1-seronegative and HIV-1-infected patients, both with and without encephalitis, revealed significant tight
junction disruption in patients who died with HIV encephalitis, as
shown by fragmentation or absence of immunoreactivity for occludin
and ZO-1 [140]. These phenomena were associated with accumulation
of activated HIV-1-infected brain macrophages, fibrinogen leakage,
and marked astrocytosis, suggesting that the main route of HIV-1infected monocyte entry into the CNS could be the disrupted BBB structure [140]. Further evidence was also reported in a study with human
brain microvascular endothelial cells where the increased BBB microvascular permeability was found to be due to the degradation of tight
junction proteins ZO-1 and ZO-2 caused by HIV type 1 gp120 [141]. In
addition to change of tight junction proteins, other alterations, such as
expression of matrix metalloproteinases in virus-infected BBB can result in the BBB integrity being compromised, allowing unrestricted
entry of immune cells into the brain, thereby contributing to virus induced neuropathogenesis [142]. A study conducted by Verma et al.
[143] indicates that a cell-free virus, such as West Nile virus, has the
ability to enter the CNS without compromising the BBB integrity. It
gained access across the BBB via the “Trojan horse” mechanism through
induction and increased expression of cell adhesion molecules that
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permit migration of monocytes/macrophages through the BBB. Recently, Foust and colleagues [144] demonstrated the intravenously administered adeno-associated virus (AAV9) can efficiently target cells of the
CNS such as neonatal neurons and adult astrocytes without disruption
of the BBB. Although the precise mechanism by which AAV9 bypasses
the BBB is unclear, it was speculated that AAV9 may utilise transport
proteins, receptor mediated transcytosis, adsorptive transcytosis or
other mechanisms to cross the BBB [144]. This unique property of
AAV9 is of great interest for development of a non-invasive method to
achieve efficient gene delivery to the CNS. When Kaplitt et al. [145]
studied the delivery of glutamic acid decarboxylase (GAD) gene with
(AAVinto the subthalamic nucleus of patients with Parkinson's disease,
they found this gene therapy was well tolerated by patients and safely
improved the symptoms of patients with advanced Parkinson's disease.
The use of viruses for gene delivery to the CNS is still in its infancy, and
its long term effects and toxicity are yet to be fully understood.
5.1.2. Enhanced by chemical stimuli
There are several examples of chemical stimuli used to modulate
the TJ to increase the permeability of the BBB for drug transport
[47]. An often employed BBB opening practice is via arterial injection
of hyperosmolar solution (e.g., mannitol, arabinose). The shrinkage of
endothelial cells results in transient opening gaps between cells. One
major limitation of this approach is its invasiveness, which requires
considerable expertise [146]. Here we will highlight actions of some
pharmaceutical excipients on the BBB. For instance, oleic acid, a PKC
activator, showed reversible opening of the BBB to Evans blue albumin and γ-aminoisobutyric acid when it was given via arterial infusion in a rat model [147]. Lysophosphatidic acid was also reported
to increase TJ permeability in cultured brain endothelia cells [148]
via the activation of PKC-alpha channels which reduces caudin-5 expression and F-actin recombination [149]. Like some biological stimuli, its action is also rapid, dose-dependent and reversible, and its
effect can be attenuated by activation of protein kinase C [148].
One of the most commonly used pharmaceutical excipients, sodium
dodecyl sulphate (SDS) has also been shown to induce an extensive but
reversible, dose-dependent Evans blue extravasations and increased
transport of α-aminoisobutyric acid at a dose of 25–100 μg/kg in rats
[150]. This is not surprising considering SDS is an anionic surfactant
with function as a solubilisation agent and may interact with lipids or
protein in the cell membrane [151].
Cyclodextrins (CDs) are cyclic oligosaccharides composed of 6, 7
and 8 glucose units; namely α-, β-, γ-CD, respectively. They are
known for their ability to form inclusion molecular complexes to increase the water solubility of hydrophobic drugs (guest molecules)
[152]. Monnaert and colleagues studied the endothelial permeability
and toxicity of native, methylated, and hydroxypropylated α-, β-, γ-CD
in an in vitro cell model [153]. Native α-, β-CD elicited a rapid increase
in sucrose permeability of cerebral endothelial cell monolayers which
correlated with their ability to extract phospholipids [153]. Among
those tested, γ-CD had the least toxicity. High concentrations of hydroxypropyl γ-CD and γ-CD increased doxorubicin passage through the
brain endothelia cell monolayers but at the expense of a loss of BBB integrity and decreased junctional staining of occludin [154]. This finding
suggests that oligosaccharide units are likely to be responsible for the
toxicity of CDs in the brain because of their extraction of lipophilic components of the BBB, phospholipids and, in some cases, cholesterol, which
may break down the brain endothelial cell monolayers or lead to decreased P-gp activity [155].
Interestingly, ammonium CD derivatives (monosubstituted nalkyldimethylammonium-β-CD derivatives, DMA-Cn-CDs 2 b n b 16)
showed much lower toxicity than native β-CD. In particular, DMAC12-CD always remained non-toxic even at a concentration as high as
10 mM [156]. It appears that a long alkyl chain is responsible for producing non-toxic effects by obstructing the CD cavity, therefore reducing the extraction ability of CDs for phospholipids and cholesterol. In
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Y. Chen, L. Liu / Advanced Drug Delivery Reviews 64 (2012) 640–665
addition, the masked amphiphilic structure of DMA-C12-CD was also
thought to be responsible for its permeability through the BBB. 30% of
DMA-C12-CD passed through the endothelial cells at 5 mM [156]. Unfortunately, all these studies were conducted in vitro, and there is yet no
confirmation if CDs would work in vivo for enhancing drug transport
across the BBB.
5.1.3. Enhanced by physical stimuli
The ability of energy-based physical methods, such as ultrasound,
microwave or electromagnetic fields, to open the BBB has been investigated. Extensive reviews can be found elsewhere [118,119].
Development of acoustic technology has enabled ultrasound to
become not only a diagnostic tool but also a therapeutic modality.
Focused ultrasound techniques concentrate acoustic energy in a
focal spot deep in the body with minimal effect to tissues outside
the field of focus [157]. This allows it to non-invasively induce local
biological effects deep inside the body and removes the need for surgical intervention. Hynynen et al. showed that introduction of a preformed gas bubble before focused ultrasound exposure would allow
transient opening of the BBB locally without causing acute damage
to the neurons [158] or delayed ischemia. The gas bubble not only
confines the ultrasound effect to the vasculature, but also reduces
the power needed to open the BBB, making it possible for the ultrasound to be applied through the intact skull. By combining with an
imaging device such as magnetic resonance imaging (MRI) scanner,
ultrasound becomes a non-invasive approach to open targeted regions of the BBB to permit the delivery of drugs and other therapeutic
molecules across the BBB [118].
Sheikov et al. studied the effect of focused ultrasound in a rabbit
model and revealed that at the acoustic power applied (0.55 W and
3 W), TJ opening occurred with leakage of dye and contrast matter
[159]. Characteristic ultrastructural changes of the brain microvasculature were seen under electron microscopy, however they were confined to sonicated locations. The electron microscopic data show that
sonication produced acoustic power-dependent tissue damage with
the damage by 0.55 W equivalent to categories 1–2 and a low level
of red blood cell extravasations [159]. In a separate study, sonication
with the same parameter, 1 MPa (0.55 W), induced no delayed damage to neurons for up to a month. [160]. Using goat antirabbit IgG
conjugated to 10 nm gold particles and electron microscopic analysis,
the group also demonstrated that the ultrasound induction of fenestration appeared to be rapid with fenestrate-like openings seen on
samples in 1 and 2 h after the sonication. The cytoplasmic channels
were observed in the endothelia cells and appear to be formations
for transendothelial passage [159], a phenomenon similar to that
reported in mouse brain capillaries in response to heat stress [161].
Evidence suggests that the local, reversible disruption of the BBB by
bursts of low frequency MRI-guided ultrasound enhances the brain
delivery of monoclonal antibody—Herceptin (trastuzumab) in mice
[162] and doxorubicin in rats [163]. It was suggested that the mechanisms for transport of molecules by focused ultrasound may involve
transcytosis, transendothelial openings—fenestration and channel
formation, widening of interendothelial clefts and partial opening of
TJs and free passage through the injured endothelium [159].
In a recent report [164], MRI-guided focused ultrasound facilitated
the delivery of anti-Abeta antibody, BAM-10 to Abeta plaques in targeted cortical areas following intravenous injection in a mouse AD
model. The reduction in Abeta pathology became apparent four days
post treatment [164]. It was suggested that the mechanisms for
transport of molecules by focused ultrasound may involve transcytosis, transendothelial openings—fenestration and channel formation,
widening of interendothelial clefts and partial opening of TJs and
free passage through the injured endothelium [159]. Such rapid reduction in plaque pathology indicates MRI-guided focused ultrasound
has great potential in enhancing the transport of both small and
large molecules across the BBB for delivery of a target therapy for
treatment of CNS disorders. There is an urgent need to further develop and fine tune the technology to allow the repeat application of
ultrasound bursts without producing sustained damage to the tissue
and the BBB.
Microwave energy has also been studied for drug transport into
the CNS [165]. It was reported that Chinese hamsters exposed to
low level microwave energy exhibited reversible permeability of the
BBB to horseradish peroxidase (HRP) [166]. In a separate study, microwave irradiation facilitated central effects of domperidone by altering the permeability of the BBB and increasing the entry of the
drug into the CNS [167]. Since sufficiently strong microwave energy
can lead to tissue heating, the resultant brain temperature increase
may produce increased BBB permeability. This was shown in a recent
study where neuronal albumin uptake in brain was shown to be correlated with brain temperature, with more apparent effects observed
when temperature was raised 1 °C or more [168]. Indeed, reports
show that exposure of the rat head to microwave frequencies (2.5–
3.2 GHz) leads to an increase of brain temperature above 40 °C,
which can enhance the BBB permeability to HRP [165], Evans blue
[169,170] and sodium fluorescein [171]. It is interesting to note that
when brain temperature was cooled down below 40 °C, microwave
irradiation failed to open the BBB suggesting the mechanism of barrier opening is not related to the non-thermal effect of microwaves
[165]. Stam reviewed all evidence reported with laboratory animals
and came to the conclusion that “acute, non-thermal microwave exposure does not increase BBB permeability” [119]. If that is true, one
has to be cautious about exposure to microwave at thermal levels as
it could put the brain at risk of infection [172].
Exposure to the electric magnetic field of MRI can also potentially
reach thermal levels under certain circumstances [119]. Although
there are studies carried out on assessment of the effects of MRI on
BBB permeability, the findings are inconsistent: some showed an increased BBB permeability [173,174], others showed no change
[175,176]. To date there is insufficient evidence to make a conclusion
on the impact of MRI on BBB permeability and drug transport.
There are also examples of using electromagnetic field (EMF)
pulses to induce the permeability of the BBB. Qiu et al. showed that
electromagnetic pulses induce BBB permeability via regulating protein kinase C signalling and translocation of tight junction's protein
ZO-1 [177]. When the antiretroviral agent saquinavir was loaded in
polymeric and solid lipid nanoparticles and investigated for BBB permeability under the influence of an EMF in an in vitro BBB model, enhanced transport of antiviral drug across BBB was observed. The level
of enhancement was dependent on wave shape, frequency and amplitude of EMF [178]. The study demonstrated the combination of
nanoparticle carrier and EMF strongly benefits drug transport and
could have potential clinical application to the therapy of brainrelated disease.
Overall, ultrasound as physical stimuli to transiently open the BBB
to enhance the drug transport into the brain has received significantly
more research effort than other methods. Although its mechanisms
for drug transport and sustained impact on tissue are not fully understood yet, MRI-guided focused ultrasound does have attractive features that it can provide diagnosis and treatment at the same time
and can be used to enhance not just small molecules, but also large
proteins or even particulate delivery systems such as liposomes and
nanoparticles [118]. It would appear to have the potential to be an
important drug delivery modality for treatment of CNS diseases.
In summary, the TJ opening approach is a double-edged sword. On
one hand, it provides enhanced BBB permeability to a large number
and variety of drugs, proteins, peptides, genetic materials, and even
drug delivery systems such as liposomes or nanoparticles, without
the need to modify the chemical structure of the drug. On the other
hand, opening of TJ for a drug molecule may also lead to the enhanced
passage of other molecules and unwanted substances such as pathogens through the protective barrier because this paracellular route is
Y. Chen, L. Liu / Advanced Drug Delivery Reviews 64 (2012) 640–665
651
not specific enough to exclude CNS entry of toxins and other
unwanted molecules. It is, therefore, important to be able to control
the time and duration of reversible opening of TJ and to ensure the
frequent application of stimuli will not have an impact on the BBB
and brain conditions, thus this approach can provide both efficacy
and safety for a brain therapy.
• BBB-targeted moiety (receptor or adsorptive mediated mechanism,
or uptake by monocytes or macrophages)
• Well maintained parent drug stability
• Tunable drug release profiles
• Applicable to carry small molecules, proteins, peptides or nucleic
acids
5.2. Transport system-mediated drug delivery across the blood–brain
barrier
Despite a large variety of nanocarriers developed so far, it is noteworthy that only amphiphilic molecule-formed liposomes and polymeric nanoparticles have been extensively exploited for brain drug
delivery [181]. Several such systems are now in clinical trials for anticancer drug delivery. For instance, the University of Regensburg in
collaboration with Essex Pharma (Schering-Plough) Germany has
completed a phase II clinical trial of pegylated liposomal doxorubicin
and prolonged temozolomide in combination with radiotherapy in
newly diagnosed glioblastoma [182]. Compound 2B3-101, a brain targeted doxorubicin liposomes coated with the tripeptide glutathione
at the tips of polyethylene glycol (PEG), is in the Phase I/II clinical
trial in the Antoni van Leeuwenhoek hospital, the Netherlands. Nonamphiphilic colloidal drug carrier systems such as dendrimers and
microemulsions are still at relatively early development stage and
therefore will not be covered in this review. In the following sections
the structure and compositions of liposomes and micelles will be
briefly introduced with emphasis on polymeric nanoparticles for
drug delivery to the brain.
The combination of endogenous transport systems present in the
BBB and macromolecular conjugates or surface enhanced nanoparticulate delivery systems such as liposomes, nanoparticles and
supermolecular complexes has been utilised to access the brain. As
discussed previously, transporter proteins, specific receptors or adsorptive endocytosis can be used to realize drug delivery. The characteristics of these systems are
1. the drug is either chemically modified, for instance, conjugated to
a ligand or polymer as a homing device thereby masking its intrinsic properties;
2. the drug is encapsulated in a surface modified drug delivery system, such as liposomes, nanoparticles or niosomes. The surface of
the drug delivery system is often modified to contain a homing device and a hydrophilic polymer such as PEG to prolong the circulation time of the delivery system. The latter is necessary for
achieving a high drug concentration gradient at the BBB [29];
3. the homing device and drug delivery system should be nonimmunogenic (unless it is targeting the monocytes/macrophages)
and capable of interacting with receptors presented at the BBB to
facilitate the uptake of the drug by the BBB;
4. the homing device must be receptor specific, thereby reducing potential side-effects and increasing transport efficiency;
5. all systems must have controlled size, therefore their properties
are uniform and consistent and their biological fate can be
controlled.
The best system will be the one which can fulfil all these characteristics and have a homing device that is specific for a target which
is induced or up-regulated by the pathological conditions (see
Section 4.2).
5.2.1. Nanocarriers for brain drug delivery
Nanocarriers are an emerging class of drug delivery systems that
can be easily tailored to delivery drugs to various parts of the body,
including the brain. In the past decade, it has been attracting increasing attention for its use in transport of drug across the BBB due to the
rapid increase in our understanding of receptors and the fast development in polymer chemistry and nanotechnology. Nanocarriers are
unique because of their size and easily tailored structures due to the
material used. They can behave like macromolecules in certain circumstances but they can carry much more drug payload and are
capable of controlling drug release. They can carry a range of drugs
and their surface properties can be modified. These properties make
nanocarriers an attractive alternative for transporting drug across
the BBB.
Nanoscale drug carriers consist of particles in the size range from
10 to 1000 nm. Ideal properties of nanocarriers for drug delivery
across the BBB are listed below [179,180]:
• Nontoxic, biodegradable and biocompatible
• Particle size less than 100 nm (except if transport via monocytes or
macrophages)
• Stable in blood (no aggregation and dissociation)
• Prolonged blood circulation time
• Non-immunogenic
5.2.2. Supramolecular architectures of aggregated amphiphilic molecules
An amphiphile, by definition, is a chemical species having a
“polar” (hydrophilic) head group and “hydrophobic” tails. Amphiphiles interact very strongly with water. At low concentration, amphiphiles accumulate at the surface of the water because the free
energy of the air-water interface is reduced. The head group of the
amphiphile orients to the water, and the hydrophobic tail group is adjacent to the air. Above a certain critical concentration the air-water
interface is saturated and the amphiphilic molecules aggregate in
the bulk of the water forming various morphologies, such as lamellar
bilayer, micelles, rods, vesicles or larger hexagonal aggregates.
Vesicles are hollow, lamellar spherical structures. In cell biology,
vesicles are relatively small and enclosed compartments, separated
from the cytosol by at least one lipid bilayer. Their size ranges from
20 nm to 100 μm, whereas the thickness of the membranes is around
3.5 nm. Liposomes are phospholipids vesicles frequently used for
drug delivery purposes [183,184]. Niosomes are non-phospholipidbased synthetic vesicles/micelles that have properties and functions
like liposomes [185,186].
Micelles are basically spherical aggregates of amphiphilic molecules dispersing in water with their hydrophilic head groups on the
surface of the sphere, and their hydrophobic tails collected inside.
An important property of micelles is their ability to increase the solubility and bioavailability of poorly soluble pharmaceuticals. The amphiphilic molecules in micelles are in constant exchange with those
in the bulk solution. On the other hand, polymeric micelles, also
known as polymersomes, are self-assembled polymer shells composed of block copolymer amphiphiles [187] such as polyethylene
glycol-polylactic acid (PEG-PLA) and PEG-polycaprolactone (PEG-PCL).
Block copolymers have the same basic amphiphilic property as lipids
(Fig. 3) but they consist of distinct polymer chains covalently linked in
a series of two or more segments [187]. Polymeric micelles differ from
nanoparticles that are either more solid or monolithic (nanospheres)
or contain an oily or aqueous core and are surrounded by a polymer
shell (nanocapsules). However, in practice, polymeric micelles also be
referred to as nanoparticle or nanocarriers because of their particle size.
Amphiphilic block copolymers have recently emerged as a special
class of materials. This is because polymer molecular weights can be
orders of magnitude greater than those of lipids, self-assembled
structures of which are rather stable. In addition, the diversity of
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Y. Chen, L. Liu / Advanced Drug Delivery Reviews 64 (2012) 640–665
Fig. 3. (a) Natural lipid versus synthetic polymer assemblies. (b) Self-assembled block copolymers from hydrated films (polymeric micelles). (c) Block copolymer labelled with fluorescent dyes and hydrated (fluoro-polymeric micelles). (d) Cryogenic transmission electron microscopy of approximately 100-nm polymeric micelles. The two arrows point to
spherical and rod-like micelles that sometimes coexist with polymeric micelles.
Reproduced from reference [187].
block composition and block length offers the possibility of designing
the most desirable drug delivery systems.
The most commonly used hydrophobic core-forming polymers are
poly(propylene glycol), poly(D,L-lactide) and poly(caprolactone).
Cholesterol is often included in membrane phospholipids to stabilize
the membrane and reduce the membrane permeability towards encapsulated materials. In this context, cholesterol is an intriguing component of cell membranes because it both toughens and fluidizes the
membrane. Cholesterol is often employed as the hydrophobic segment because cholesterol possesses good bio-compatibility and the
potential for interaction with cholesterol receptors on the cell surface,
and strong ability to drive self-assembly of cholesterol containing
polymers [188].
5.2.3. PEGylation of nanocarriers
Most studied brain-targeted systems are liposomes, polymeric micelles and nanoparticles. These systems are able to protect enclosed
drug from chemical and biological degradation in the blood stream,
control their release, permit surface modification with targeting ligands and allow for steric stabilisation or PEGylation. Liposomes are
made of natural biological lipids with structural similarity to the cell
membrane and are therefore considered to be biologically compatible
with low toxicity [189]. Without the use of surface modification by a
hydrophilic polymer such as polyethylene glycol (PEG), the biological
half-lives of liposomes is very short due to a number of factors including the tendency of the liposome to exchange lipid materials with cell
membranes and their uptake by phagocytes [189]. The grafting of PEG
to the surface of liposomes significantly changed their fate and extended their residence time in vivo. Thus PEGylated liposomes have
become a standard preparation particularly when tissue/receptor targeting is desired [190–192]. A highly flexible and hydrated PEG chain
attached to the liposomes surface is assumed to have an effective
opsonins-resistant property due to its steric repulsion effect [189].
The PEGylation or stealth technology has also been applied to all
other drug delivery systems, including macromolecular conjugates,
nanoparticles, dendrimers and polymeric micelles [187]. Grafting
PEG to polymer or including PEG as part of the block copolymers essentially also made the polymer molecules amphiphilic.
PEG-containing surfactants, poly (oxy-ethylene)-poly(oxy-propylene) block copolymers, (poloxamer 338 = Pluronic®F108 and
poloxamine 908 = Tetronic ®) were also found to be effective in
prolonging the blood circulation time of nanoparticles, even by coating [193,194].
Compared to PEG-surfactant coating micelles or nanoparticles,
self-assembly from PEGylated polymers provides a better choice due
to the chemical binding of PEG onto the nanoparticles. The PEG chains
were first bound to poly(lactic acid) nanoparticles by Gref et al. [195]
and Bazile et al. [196]. Later PEG was chemically linked to poly(hexadecyl cyanoacrylate (PHDCA) nanoparticles [197]. Both PEGylated
poly(lactic acid) and PHDCA nanoparticles, significantly prolonged
the blood circulation time and reduced liver uptake. Calvo et al. investigated the distribution of PEG-PHDCA nanoparticles in EAE rats
[198]. Analysis by confocal microscopy showed evidence that fluorescent PEG-PHDCA nanoparticles were present in the epithelial cells of
the pia mater, ventricles, spinal cord surface and in the ependymal
cells of the choroid plexus, with less found in the endothelia cells of
the BBB. In the same study, Poloxamine 908-coated PHDCA and
polysorbate-80 coated nanoparticles exhibited less penetration of
the BBB. It was hypothesized that PEG-PHDCA nanoparticles reached
the brain by two mechanisms: passive diffusion due to the increase of
the BBB permeability and transport by nanoparticles-containing macrophages, which infiltrated these inflammatory tissues. The PEGylated PHDCA nanoparticles accumulated in the brain at a 4–8 fold
higher concentration than the non-PEGylated PHDCA nanoparticles
Y. Chen, L. Liu / Advanced Drug Delivery Reviews 64 (2012) 640–665
653
after intravenous injection into rats with 9 L gliosarcoma [199].
Therefore, the covalent attachment of PEG chains was a promising
step forward. Other PEG-containing copolymers showing potential
ability for drug delivery to the brain are depicted in Fig. 4.
PEG corona thus excluding steric hindrance for their binding to the
target receptors [29].
5.2.4. Functionalization of nanocarriers for drug transport across the
blood–brain barrier
Functionalization of nanocarriers is one of the most important
steps or challenges in formulating nanocarriers for drug delivery.
The challenge is not only in the chemistry, but also in selecting and
designing appropriate targeting ligands that can achieve the targeting
or homing function.
Functionalization itself entails a thorough understanding of the
target organ and transport mechanisms, and more importantly their
functions in relation to a particular pathological condition, which
was discussed previously. As shown in Tables 1 and 2, some of the
transport mechanisms may be up regulated or down regulated
depending on the type of brain disease. There are also a number of
agents which can have direct effect on the BBB (Table 2) [180]. One
needs to take into consideration the sensitivity of the BBB to the functional groups on the surface of the nanocarrier as well as the potential
dose dependent toxicity of the ligands.
In terms of a chemical approach to functionalization, PEG is a good
candidate for functionalization. In addition to providing steric stabilisation, the presence of PEG on the surface of the nanocarriers also allows for the preparation of bioactive nanocarriers by attaching
bioactive ligands on the surface for targeted delivery to the brain.
There are obvious reasons for the design of ligand coated longcirculating drug carriers:
One of the successful strategies of the delivery of molecules that
are unable to pass the BBB is the use of transport vectors which activate natural transport routes. The endogenous carrier-mediated
transport (CMT) for nutrients and AMT for peptides can be portals
of entry to the brain for circulating drugs.
The BBB expresses several transport systems for nutrients [200],
but the utilisation of these transport systems for targeting drugs
into the brain is mainly limited to peptide drugs, which must have a
molecular structure mimicking the endogenous nutrients. The prototypical example is levodopa, a lipid-insoluble precursor of dopamine
that has been used for the treatment of Parkinson's disease because
it contains the carboxyl and α-amino groups that allow it to compete
for transport across the BBB by the large neutral amino acid carrier
[201]. In addition, since nutrient carriers stay in the membrane of
the cell, the size of the drugs must be close to that of the endogenous
ligand if they need to be taken up and transferred into the brain [200].
This approach is generally less favoured because it may interfere with
the transport of nutrients and also for certain molecules, for instance,
antibiotics, that do not have structures that are close to that of endogenous ligands.
1) Ligand (an antibody, protein, peptide, sugar moiety, folate or carbohydrate) attached to the carrier surface may increase the rate of
elimination from the blood and uptake in the liver and spleen.
However, the presence of the PEG protecting polymer may compensate for this effect;
2) Longevity of the specific ligand-bearing nanocarrier may allow for
its successful accumulation in targets with diminished blood flow
or with low concentration of the surface antigen.
Covalent linkage of targeting ligands to the nanocarriers is typically made by a simple coupling reaction between amine-functionalized
nanocarriers and succinimidyl ester derivatives. To achieve better selective targeting, ligands should be attached to the nanocarrier via the
PEG spacer arm, so that the ligands are extended outside of the dense
Fig. 4. Selected examples of PEG containing block polymers which can be selfassembled to form polymeric micelles for drug delivery.
5.3. Drug transport across the blood–brain barrier via transport vectors
5.4. Drug transport across the blood–brain barrier via
adsorptive-mediated transcytosis
AMT has recently gained significant importance as a route for drug
delivery into the brain due to growing evidence showing the success
of this transport route for delivery of drugs into the brain via cationic
proteins, and cell-penetrating peptides (CPPs) [24]. CPPs are positively charged peptides with amphipathic characteristics. They are capable of rapidly entering living cells without producing cytolytic effects.
They have been successfully used as vectors for delivery of drugs
that are P-gp substrates by effectively by-passing the P-gp in the
BBB. For instance, they have increased doxorubicin transport into
the rat brain up to 30-fold [202]. This approach is effective because
cell-penetrating peptides utilise adsorptive-mediated endocytosis to
enter the brain. Another notable success is the use of vectors such
as SynB3 (RRLSYSRRRF) to increase brain uptake of poorly brainpenetrating drugs [24,203]. SynB peptides derive from a natural
mammalian antimicrobial peptide with high affinity for biological
membranes. It is one class of CPPs that have emerged, facilitating
the intracellular delivery of polar biomolecules in vitro and in vivo.
It was shown to enhance the transport of morphine-6-glucuronide
to the brain in a clinical trial [30].
CPPs refer to a group of short peptides of less than 30 amino acids
that are able to penetrate cell membranes and transport their cargo
into cells [204]. Table 3 summarises the majority of CPPs with their
principal features. While these individual CPPs differ in length and sequence, they share a few common features, which include their amphipathic nature, net positive charge, theoretical hydrophobicity
and helical moment, the ability to interact with lipidic membranes,
and to adopt a distinct secondary structure upon association with
lipids [205]. The major dogma has been that CPPs enter cells by a receptor and energy-independent process but the exact mechanisms
are not yet fully understood. For some CPPs, endocytosis is an exclusive and alternative mechanism of internalization. For instance,
SynB's internalization is a temperature and energy dependent process
and involves endosomal transport. It has been confirmed that SynB
penetrates into cells via AMT [206]. On the other hand, TAT (HIV-1
trans-activating transcriptor) derived CPPs enter cells primarily by
lipid raft-mediated macropinocytosis that is stimulated by cellsurface binding of TAT derived CPPs [207].
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Table 3
Principle features of the selected cell penetrating peptides (CPPs).
Peptide name
Sequence
Net
charge
Cell lytic
activity
MAP
pAntp43–68
Transportan
SBP
FBP
TAT48–60
SynB1
SynB3
KLALKLALKALKAALKLA
RQIKIWFQNRRMKWKK
GWTLNSAGYLLGKINLKALAALAKKIL
MGLGLHLLVLAAALQGAWSQPKKKRKV
GALFLGWLGAAGSTMGAWSQPKKKRKV
GRKKRRQRRRPPQ
RGGRLSYSRRRFSTSTGR
RRLSYSRRRF
+5
+8
+4
+6
+6
+8
+6
+6
Yes
No
Yes
–
–
No
No
No
MAP: model amphipathic peptide; Antp: Antennapedia; SBP: sequence signal-based
peptide; FBP, fusion sequence-based peptide; TAT, HIV-1 trans-activating transcriptor.
The peptide residues in this table are expressed with one-letter-code: K—lysine; L—
leucine; A—alanine; R—arginines; Q—glutamine; I—isoleucine; W—tryptophan; F—
phenylalanine; N—asparagine; M—methionine; G—glycines; S—serine; T—threonine. Data
was collected from references [204,206,221,222,312–314].
The application of CPPs is based on the premises that biologically
active cargo can be attached to CPPs and translocated into cells. The
link between the CPPs and cargo is most commonly a covalent bond
and seldom in non-covalent bond. A large variety of cargo molecules/materials have been effectively delivered into cells via CPPs, including small molecules, proteins, peptides, fragments of DNA,
liposomes and nanoparticles [204]. Some can enter brain capillary endothelia cells or are even translocated into the brain tissues. Some examples are highlighted here.
Adenot and colleagues studied brain uptake of a number of free
and SynB3 vectorized chemotherapeutic agents using both in situ
brain perfusion and in vitro BBB/cell model [203]. They reported
that SynB3's conjugation with various poorly brain-penetrating
drugs enhanced their brain penetration by a factor of 30 for doxorubicin, 7 for benzylpenicillin, 22 for paclitaxel, 18 for dalargin and 50 for
morphine-6-glucuronide with no effect on tight junction integrity.
Brain uptake of the enkaphalin analogue, dalargin, a hexapeptide,
was enhanced significantly when conjugated to SynB and injected intravenously in mice [208]. This study signalled the potential for delivery of peptides or drugs for treatment of brain cancer, through the
targeting of brain tissue after systemic delivery.
TAT is a HIV-1 trans-activating transcriptor with 101 amino acids.
The protein consists of five domains; probably the best-studied region
of TAT is located in domain 4, which contains a highly basic region
(with two lysines and six arginines in nine residues) involved in nuclear
and nucleolar localization [209]. While all CPPs listed in Table 3 above
have been used mainly for small cargoes such as peptides and oligonucleotides, Schwarze et al. [210] synthesized full-length fusion proteins
that contained a NH2-terminal 11-amino acid protein transduction domain (PTD) from the HIV TAT protein. Transduction of the proteins
evaluated was non-cell-specific, and was seen to occur even across
the BBB. Further proof of this mode of peptide delivery was attained
by Cao et al. [211] who fused the antiapoptotic protein Bcl-xL to TAT
and injected the construct intraperitoneally into mice that were affected by stroke. The Bcl-xL protein is expressed in adult neurons of the CNS
and is believed to have an important role in the prevention of neuronal
apoptosis that would normally occur during brain development, or results from varying stimuli leading to pathology, including cerebral
ischemia. Protein transduction with this entity occurred in a rapid,
concentration-dependent fashion, with entry into cells thought to
occur via the lipid bilayer component of the cellular membrane. A
study by Kilic et al. [212] using the same model showed that brain tissue
was progressively transduced with TAT proteins within 3–4 h after intravenous delivery. TAT-Bcl-xL treatment reduced infarct volume and
neurological deficits after long ischemic insults lasting 90 min, when
applied both before and after ischemia.
Studies have also shown that even relatively large particles could be
delivered into various cells by TAT vector. A biocompatible 45 nm
nanoparticle with an iron core, a dextran coating, and covalently linked
TAT peptides was efficiently taken up by human hematopoietic CD34+
cells [213]. Even cytoplasmatic uptake of liposomes with a diameter of
200 nm has been documented [214].
Taking the TAT-mediated nanoparticles delivery approach a step
further, one of the most exciting demonstrations of the effectiveness
of TAT-shuttled nanocarriers across the BBB was accomplished by
TAT-conjugated CdS:Mn/ZnS quantum dots (Qdots) [215]. Histological data clearly showed that TAT-Qdots migrated beyond endothelial
cells and reached the brain parenchyma. TAT-mediated intracellular
delivery of large molecules and nanoparticles was proved to proceed
via the energy-dependent macropinocytosis with subsequent enhanced escape from endosome into the cell cytoplasm [207]. Recently, Liu et al. produced compelling evidence that TAT facilitates human
brain endothelia cell uptake of nanoparticles self-assembled from
TAT-PEG-b-cholesterol in vitro and more importantly, the nanoparticles with TAT were able to cross the BBB and translocate around the
cell nucleus of neurons [216]. This study demonstrates the effectiveness of TAT in promoting the transport of nanoparticles across the
BBB. It confirms that nanocarriers conjugated with TAT could be a
promising carrier system for transporting drug across the BBB for
the treatment of brain disorders.
In a more recent study by Wang et al., cationic nanoparticles fabricated from cholesterol-CG3R6TAT via self-assembly showed strong antimicrobial activity [217]. Biodistribution studies of FITC-loaded
nanoparticles in rabbits and efficacy studies in a C. neoformans meningitis rabbit model revealed that these nanoparticles crossed the BBB and
produced antimicrobial activity against the pathological strains in the
brain tissue with a similar efficacy as amphotericin B, suggesting a therapeutic dose was delivered by TAT containing nanoparticles. Furthermore, these nanoparticles avoided causing the side-effects associated
with amphotericin. This study holds importance for TAT-containing
nanoparticles as it has proven that it is possible to deliver a therapeutic
dose, together with functional agents, via TAT-nanoparticles into the
brain for treatment of brain infections and tracking of nanoparticles in
vivo, a step closer to the development of a clinically applicable nanocarriers for treatment as well as monitoring meningitis and other brainrelated disorders.
Recent evidence showed that TAT can also enhance the delivery of
liposomes into the brain. Qin et al. prepared liposomes using
cholesterol-PEG2000-TAT (TAT-LIP) and compared them to liposomes
fabricated from cholesterol-PEG2000 polymer (LLIP) and conventional
cholesterol formulation (LIP) in vitro and in vivo [218]. TAT-LIP accumulated most in the brain (including various regions of the brain)
within 24 hr after administration via tail vein, although all were not
selectively targeted to the brain. All liposomes showed a uniform distribution across the brain. The study also suggested adsorptive transcytosis could be one of the mechanisms for TAT-LIP transport across
the BBB and the positive charge of the TAT-LIP played an important
role in enhancing this transport [218].
In addition to CPPs, cationic protein can also enter the brain via an
adsorptive-mediated mechanism and Poduslo and Curran demonstrated that polyamine modification of proteins (insulin, albumin
and IgG) can dramatically increase the permeability of proteins at
the BBB with 1.7–2.0 fold increase for insulin, 54–165 folds for albumin and 111–349 fold for IgG in normal adult rats [219]. It is, however, unknown, if this chemical modification may lead to toxicity or
immunogenicity problems. In a study reported by Lu et al., cationic
bovine serum albumin (CBSA) conjugated PEG-PLA nanoparticles
(CBSA-NP) was compared to native PLA bovine serum albumin conjugated nanoparticles (BSA-NP) and CBSA unconjugated PEGylated
nanoparticles (NP) in brain transcytosis across the BBB coculture
and brain delivery in mice using a fluorescent probe [220]. This
study confirmed that AMT is the mechanism of brain delivery of
CBSA-NP. Increasing the surface density of CBSA conjugated per
nanoparticle promoted the transcytosis ability of nanoparticles
Y. Chen, L. Liu / Advanced Drug Delivery Reviews 64 (2012) 640–665
while their blood AUC decreased. With the optimized CBSA number
conjugated per nanoparticle of 1:10, CBSA-NP achieved the highest
% injection dose/g brain by 2.3-fold compared with NP [220]. The
same study also reported an accelerated blood clearance phenomena
can be induced by first injection of NP or CSBA-NP or over a period of
successive high dose of CBSA-NP, highlighting the importance of the
issue and potential related toxicity and immunological responses
unique to this type of nanocarrier.
AMT enables many poorly brain-penetrating drugs across the BBB,
and holds potential for promoting drug delivery into the brain. However, because it is a non-specific process, the adsorptive process will
not only occur at the BBB but also in the blood vessels in other organs.
This poses a challenge for both achieving therapeutic concentration in
the brain and limiting the drug distribution in non-target organ. Toxicity and immunogenicity issues, especially with cationic proteins,
should not be overlooked and must be assessed for each system.
There are studies that can be carried out to assess both the membrane
toxicity and tissue inflammation caused by above mentioned CPPs
and cationic albumin nanoparticles [220,221]. Some membrane toxicity studies have indicated that cytotoxicity of different CPPs can vary.
For instance, TAT is non toxic with concentration up to 100 μM,
whereas Antp is significantly more toxic [222]. Furthermore, peptides
bound to TAT can trigger significant and chain length- dependent cytotoxicity when their concentration is above 10 μM, irrespective of
the sequence of cargo [222]. In addition, a biocompatibility and
dose-dependent cytotoxicity study should be carried out. For instance, the basic domain TAT49–57 has been used in inhibiting neural
death [223] and achieving biological effects by delivering large proteins, such as RNase, domain III of pseudomonas exotoxin A, horseradish peroxidase and β-galactosidase [224] into the intracellular
space [225]. A very high dose of TAT46–60 peptide was reported to
be toxic ([226]. The cytotoxicity of TAT-conjugated nano-carrier
should be assessed against human brain microvascular endothelial
cells and astrocytes. Cell viability could be measured by following a
standard MTT assay procedure. The effect of the degree of TAT conjugation should also be investigated and optimized to gain an understanding of the toxicity of various carrier systems and their
structure and dose relationship.
5.5. Drug transport across the blood–brain barrier via endogenous
receptor-mediated transcytosis
Despite the huge success with some AMT based drug delivery systems, one of the biggest shortcomings of AMT is its lack of selectivity,
which potentially can cause side effects of drugs in non-targeted organs. On the other hand, RMT provides an opportunity for active targeting of BBB, particularly when the target receptor is up-graded
under the diseased condition, e.g. diphtheria toxin receptor under inflammatory disease conditions. Cargo molecules which are associated
with targeting ligands can be transported across the BBB via this approach, therefore, RMT is also known as the molecular Trojan horse
approach [227,2]. Although this approach was originally applied to
molecular or macromolecular cargos [228,229], it has now been extended to transport drug-carrying nanocarriers with the same level
of success (see below).
The progress in molecular biological and BBB genomics enables
the rapid discovery of novel transporters that are expressed at the
brain capillary endothelium, potentially a large number of receptors/transporters expressed at the BBB can be utilised for drug delivery across the BBB into the brain. In this section, focus will be on those
most studied and potentially most effective systems.
In general, there are three steps for RMT: 1) endocytosis at the luminal (blood) side after receptor-ligand binding; 2) movement
through the endothelia cytoplasm; 3) exocytosis of the drug or
ligand-attached drug or cargo at the abluminal (brain) side [227].
Step two however, may involve endosomal/lysosomal systems,
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which potentially can degrade drug molecules, therapeutic protein
and peptides and genetic materials. To escape this fate, pH-sensitive
liposomes or use of cationic molecules have been applied [230,231].
Some molecules, such as diphtheria toxin, when used as a targeting
ligand, have intrinsic lysosomal escape mechanisms [232]. Fortunately, lysosomal escape mechanism is not essential in brain delivery,
RMT has been successful in transporting large drug molecules, drugcarrying liposomes, nanoparticles and polymeric complexes to the
brain even without it [30,233].
5.5.1. Insulin receptor
The insulin receptor is a large protein having a molecular weight of
300 kDa and is a heterotetramer of two extracellular alpha and two
transmembrane beta subunits [30]. Each beta chain contains a tyrosine
kinase activity in its cytosolic extension. This is a widely characterised
receptor-mediated transcytosis system used for transporting drugs
and genes into the brain. Extensive studies of the insulin-receptor and
its uses have been carried out by Pardridge and colleagues [234–237].
Pardridge's group successfully developed a radiolabelled amyloid-βpeptide, 125I-Abeta1–40 conjugated to 83–14 monoclonal antibody
(mAb) that binds to the human insulin receptor as a diagnostic probe
for AD [238]. Recently, the same group tested a genetically engineered
human/mouse chimeric form of the human insulin receptor monoclonal antibody (HIRMAb) on an adult anesthetized Rhesus monkey and
showed that humanized HIRMAb was rapidly transported into all
parts of the primate brain after intravenous administration, suggesting
its potential for delivering drug and gene across the BBB in human
[239]. However, this approach is still considered as a risky one as it
targets one of the most important mechanism involved in glucose
homeostasis.
5.5.2. Transferrin receptor
The most widely characterised RMT system for drug delivery across
the BBB is the TfR, even though the intracellular trafficking of transferrin
upon internalization via the TfR has not yet been elucidated. The TfR is a
transmembrane glycoprotein consisting of two subunits of 90 kDa
linked by a disulfide bridge. Each subunit can bind one transferrin molecule [240]. The TfR is not unique to the BBB, it is also expressed on hepatocytes (mostly), erythrocytes, intestinal cells, monocytes in addition
to endothelial cells of the BBB. In the brain, the TfR can also be found on
choroid plexus epithelial cells and neurons. The TfR mediates cellular
uptake of iron bound to transferrin.
There are two ways of utilising TfR for transporting drugs into the
brain: using endogenous transferrin as targeting ligand or an antibody
directed against the TfR (e.g. OX-26). The latter binds to a different
site from that of transferrin, therefore, it is less likely to be affected
by or interfere with endogenous transferrin. da Cruz et al. [241]
reported that cationic liposomes decorated with transferrin resulted
in a significant enhancement of luciferase gene expression activity in
C6 glioma cells, primary hippocampal neurons and primary cortical
neurons. However, the transfection efficiency of this system was low.
The use of transferrin as a delivery vector may not be advantageous
due to the very high concentration of endogenous transferrin in the circulation, which competes for BBB transferrin binding sites. The likelihood
of overdosing with iron, when one tries to displace the endogenous
transferrin with exogenously applied transferrin-containing systems, is
another limiting factor for TfR in vivo application.
To overcome the limitations of using transferrin as the targeting
ligand in vivo, antibodies binding to an epitope of the TfR that is different to the transferring binding site, have been developed. OX26
(anti-rat TfR monoclonal antibody), R17-217 and 8D3 (both antimouse TfR monoclonal antibody) have all been examined. When R17217 was compared to 8D3, the brain uptake of R17-217 was relatively
low at 1.7% injected dose/g, whereas, 8D3 was 3.1% injected dose/g
[242]. However, Lee et al. found that in contrast to 8D3, R17-217 was
more selective for the brain and poorly taken up by the liver and kidney
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[242]. Ulbrich et al. studied human serum albumin (HAS) nanoparticles
with covalently coupled transferrin or transferrin receptor monoclonal
antibodies (OX26 or R17-217) for brain delivery of loperamide, a molecule that is normally unable to cross the BBB on its own [243]. Results
showed that significant anti-nociceptive effects were detected with
loperamide-loaded HAS nanoparticles with covalently bound transferrin or the OX20 or R17-217 antibodies following iv injection. A similar
efficiency in delivery of the drug into the brain was achieved with the
three different types of nanoparticles but not with the IgG2a-modified
HAS nanoparticles [243], suggesting that OX26 or R17-217 can be a
good alternative to transferrin for transporting drugs across the BBB
with similar efficiency.
In a more recent study conducted by Rooy et al. [244], five different targeting ligands: transferrin, R17-217 (against TfR), COG 133
(against LDLR and LRP), Angioprep-2 (against LRP) and CRM197
(against DTR) were directly compared for their ability to target the liposomes to the brain in vitro and in vivo. Only R17-217 and CRM197
attached liposomes were found to be associated with human endothelia cells in vitro and only R17-217 significantly enhanced the
brain uptake of liposomes in vivo at all time points after tail vein injection to male Balb/c mice [244]. Using the brain capillary depletion
method, the authors determined the uptake of 3H-labelled liposomes
in brain capillaries and found R17-217 liposomes were 10 times
higher than untargeted liposomes. The biodistribution study showed
the uptake of R17-217-liposomes was 4.3 times higher than the control 6 h post injection [244]. Interestingly R17-217 liposome is the
only one whose concentration in the brain was maintained over the
period of 6 h and its dose in the brain at 12 h was 0.18%/g. Authors
suggested that the high molecular weight (i.e. longer chain and protrude more from the surface) and high affinity for the receptor may
have contributed to its strong brain targeting ability [244]. Although
targeting ligand is an important factor in determining the performance of a drug delivery system, other parameters may also influence
the fate of the system, such as the matrix material, particle size, surface properties, the density and conformation of targeting ligand.
More studies are needed to fully understand the function and performance of the targeting ligands on different types of nanocarriers.
5.5.3. Low-density lipoprotein receptor related proteins 1 and 2 (LRP1
and LRP2 receptors)
LRP1 and 2 are multifunctional, multi-ligand scavenger and signalling receptors. Together, they can interact with a diverse range of
molecules and mediators including ApoE, tissue plasminogen activator (tPA), plasminogen activator inhibitor 1 (PAI-1), amyloid precursor protein (APP), lactoferrin, melanotransferrin, α2 macroglobulin
(α2 M), receptor associated protein (RAP), HIV-1 TAT protein, Heparin cofactor II, heat shock protein 96 (HSP-96) and engineered angiopeps [30,233]. Over the years, the low-density lipoprotein receptor
related protein-1 (LRP1) and LRP2 receptors have been exploited to
target drugs to the brain in a fashion similar to that used for the transferrin and insulin receptors. There are some successful examples
using LRP1 and LRP2 receptors for brain drug transport.
Polysorbate 80 coated poly(butylcyanoacrylate) (PBCA) nanoparticles were first investigated [245] in vivo to enhance the brain transport of hexapeptide dalargin. Results showed that after i.v. injection,
the polysorbate 80 coating enabled the highest induction of analgesia
in mice. Likewise, other drugs that normally do not penetrate the
BBB, including tubocurarine, loperamide, 8-chloro-4-hydroxy-1oxol, 2-dihydropyridazino, quinoline-5-oxide choline salt (MRZ 2/576),
and doxorubicin showed higher concentrations in the brain when associated with polysorbate 80-coated nanoparticles [246]. The protein
ApoE or B absorption on the nanoparticles, followed by low density
lipoprotein (LDL) receptor mediated endocytosis and transcytosis, is believed to facilitate the uptake of the nanoparticles by the brain. Recently
Zensi reported that PEGylated albumin nanoparticles with covalently
bonded ApoE showed enhanced uptake and intracellular localization in
mouse endothelia cells [247]. More importantly, following administration into the jugular vein of SV129 mice, the ApoE albumin nanoparticles
transcytosed into brain parenchyma and were taken into the neurones of
the mice, as supported by micrographs of different brain regions taken
30 min post injection [248]. This study is one of a few investigations
used Transmission Electron Microscopy (TEM) to provide direct high
quality visual evidence of transcytosis of ApoE albumin nanoparticles
both in vitro and in vivo.
Lactoferrin (Lf) is a mammalian cationic iron-binding glycoprotein
belonging to the transferrin family. It is an interesting molecule as it
has been implicated in the pathogenesis of brain lesions. An elevated
level of Lf has been found to be associated with normal ageing and to
be more pronounced in people with AD, Guamanian cases, Pick's disease, and Down syndrome [249]. Lf was reported to be transported
into the brain via LRP mediated transcytosis by some researchers
[250] and others showed evidence to support that there are Lf
receptors on brain capillary endothelia cells in mouse at least [251].
Fillebeen et al. demonstrated in a bovine brain capillary endothelial
cell model that when the inflammatory mediator TNF-α was present,
Lf transport in vitro through the brain capillary endothelia cells was
markedly decreased [82]. This suggests that the transport of Lf could
be different under the pathological condition compared to that of
the normal condition. The reduction in Lf transport under the inflammatory condition may work in favour for drug transport as more Lf
receptors would be free for exogenous Lf from the drug delivery
systems.
Interestingly, Ji et al. demonstrated that the brain uptake of Lf in
rats was much greater than that of transferrin and OX26 [252].
When Lf-conjugated PEG-PLA nanoparticles were administrated to
mice intravenously, there was a 3-fold increase in brain uptake of
Lf-nanoparticles compared to un-conjugated nanoparticles [253].
The cell viability study also indicated that it was non-toxic [253].
Lately, the same group assessed biodistribution of coumarin-6loaded Lf-conjugated PEG-PLA nanoparticles in mice and therapeutic
efficacy of urocortin-loaded Lf nanoparticles on a 6-OHDA rat model
of Parkinson's disease [254]. Their data showed 2.5 times increase in
AUC by Lf nanoparticles compared to the un-conjugated nanoparticles in 24 h. Drug-loaded Lf- nanoparticles were successfully taken
up by the brain and produced therapeutic efficacy, as demonstrated
by the significant attenuation of striatum lesion despite the drug
loading being only 0.28–0.33% [255]. This compelling evidence suggests that Lf-nanoparticles could be a promising drug delivery system
for treatment of Parkinson's disease if their safety in human can be
confirmed.
Another interesting targeting molecule is melanotransferrin (P97)
which has been reported to undergo active transcytosis, possibly via
the LRP1 receptor [256]. It was revealed that recombinant human
melanotransferrin (P97) was readily taken up by mouse brain following intravenous injection and in situ brain perfusion [256]. This P97
transcytosis across the bovine brain capillary endothelial cell monolayers was at least 14-fold higher than that of transferrin, with no apparent intra-endothelial degradation. When the effectiveness of P97
as a brain targeting ligand was tested in a mouse model, P97 and
P97-adrimycin conjugates showed 6–8-fold higher than that of BSA
or lactoferrin in terms of transport into the brain. More importantly
their efficacy against intracranial rat C6 glioma and human ZR-75-1
mammary tumours in athymic mice was very significant with improved survival rates compared to free adriamycin [257]. This technology has now been patented worldwide and the technology
(NeroTrans TM transporter platform) is the proprietary property of
Raptor Pharmaceutical Corp (Novato, CA). In June 2009, Raptor and
Roche entered a collaboration and licensing agreement to evaluate
the delivery of Roche's investigative molecules attached to Raptor's
proprietary NeuroTrans™ transporter platform [258].
Another group of LRP ligands, known as angiopeps, has also been
reported as a highly effective BBB targeting ligand. Angiopeps belong
Y. Chen, L. Liu / Advanced Drug Delivery Reviews 64 (2012) 640–665
to a family of peptides derived from Kunitz domains of aprotinin and
other human proteins. They are known to have affinity for LRP receptors [259]. The most studied is angiopep 2 (TFFYGGSRGKRNNFKTEEY,
molecular weight 2.4 kDa) which has shown greater transcytosis
capacity and parenchymal accumulation than transferrin, lactoferrin,
and avidin [260]. Moreover, their ability in efficiently facilitating nanocarrier transport across the BBB in vivo has been confirmed with dendrimers [261] and more recently with amphotericin B-loaded
polymeric micelles [262]. Chemical conjugation of angiopep 2 with 3
molecules of paclitaxel (ANG1005) was shown to be particularly effective in enhancing drug uptake into the brain, with an 86-fold increase
compared to the free drug using an in situ rat brain perfusion model. It
also exceeded the free drug by 4–54-fold following i.v injection in
mice bearing metastases of breast cancer [263]. A therapeutic amount
of ANG1005 was able to cross the BBB, as evidenced by the increased
survival rates of mice with implanted tumour cells [264].
It is interesting to note an innovative approach reported recently
by Tosi and colleagues. They developed poly(D,L-lactide-co-glycolide)
(PLGA) nanoparticles that are surface modified with two ligands: one
is a BBB-penetrating peptide (similopioid peptide, g7) for transporting across the BBB, another is a sialic acid residue (SA) for the interaction with receptors in the brain tissue to prolong the nanoparticle
residence time in the brain parenchyma [265]. The researchers
reported a remarkably high dose (6% injected dose) in the CNS over
a prolonged period of time (24 h). Surprisingly when the nanoparticles were conjugated with only g7 molecules, the brain uptake of
nanoparticles increased to 14% injected dose/g at 1.5 h post injection
via the tail vein, which was more than twice that of the nanoparticles
with double targeting ligands. The g7-nanoparticles, however, had a
much shorter opioid effect (5 h). With an additional SA ligand, the
central opioid activity of loperamide increased to 24 h [265]. These
remarkable results were attributed to the ability of SA g7 nanoparticles to cross the BBB and remain within the brain parenchyma. The
unusually high brain uptake of g7 nanoparticles suggests that g7 is a
promising BBB targeting ligand on its own. Finally, Tosi's data also reveals that the clearance of nanoparticles in the brain parenchyma is
very rapid without SA ligand and we speculate that this removal of
nanoparticles is more likely due to physical translocation rather
than enzyme degradation of nanoparticles.
5.5.4. Diphtheria toxin receptor (DTR)
DTR is also known as transmembrane HB-EGF. Uniquely, it has no
endogenous ligand. Although diphtheria toxin (DT) can bind to this
receptor and enter the cell via endocytosis [266], DT is too toxic to
be used in vivo. Cross reacting material (CRM 197) is a mutated
form of DT but without the enzymatic activity that would cause the
toxicity seen in DT. Because it is non-toxic, yet, retains its ability to
bind to DTR [267], it makes CRM197 a useful and attractive targeting
ligand. In fact, CRM 197 has been used as a safe and effective carrier
protein for human vaccines for a long time and has been tested as a
therapeutic protein for cancer treatment in a clinical trial [268,269].
CRM 197 uses DTR as its transport receptor and delivers its cargo molecules across the BBB by receptor mediated-transcytosis [99]. It has
been shown that CRM197 can achieve brain targeting in vitro and in
vivo [270,99].
One interesting feature of DTR is that it is strongly up-regulated
under inflammatory conditions [99], which occur often with CNS diseases such as AD, Parkinson's disease, MS, ischemia, encephalitis, epilepsy, tumour and lysosomal storage disease. This up-regulation of
DTR has already been seen following seizures [100]. This provides opportunities for disease-induced targeting and consequently may enhance the transport of the drug across the BBB and maximise the
therapeutic efficacy of the drug in the brain. If CRM197 is attached
to a cargo of diagnostic agent, then it could be used to image the inflammatory condition in patients. Therefore, it is possible to allow
657
both diagnosis and treatment to be delivered at the same time by a
nanocarrier system conjugated to the CRM197.
5.6. Drug transport across the blood–brain barrier via inhibition of efflux
Pumps
Efflux pumps such as P-gp and MRPs can prevent many drugs
from entering and accumulating in the brain. Evidence shows that
P-gp excludes a number of lipophilic compounds that may be structurally unrelated, from cerebral endothelial cells [271]. Many MRP
substrates are amphiphilic anions with at least one negatively
charged group, although MRP can also transport cationic and neutral
compounds. To circumvent this blockade, one strategy is to coadminister the drug with a pharmacological modulator which inhibits
efflux transport systems in brain capillary endothelia cells. The concept of using of Pluronic ® block (or poloxamers) copolymers to inhibit the P-gp efflux pump was derived from an early study conducted in
Kabanov laboratory by Miller et al. [272]. The study examined cellular
accumulation of rhodamine 123, a selective P-gp substrate in bovine
brain microvessel endothelial cell monolayers, with and without the
presence of Pluronic ® P85. Extensive studies have now confirmed
that Pluronic® P85, a Pluronic ® block copolymer, can be used not
only as an effective efflux pump inhibitor but also as a drug delivery
vehicle [273]. Pluronic® block copolymers are simple yet unique.
They consist of hydrophilic ethylene oxide (EO) and hydrophobic
propylene oxide (PO) blocks. EO and PO are arranged in a basic A-BA tri-block structure. This arrangement gives rise to their amphiphilic
character. The PO/EO ratio determines the hydrophobic- hydrophilic
balance (HLB) of the copolymers. Due to their amphiphilic nature,
these copolymers exhibit the properties of surfactants, including the
ability to accumulate and interact with hydrophobic surfaces and biological membranes. When their concentration in water is above
the critical micelle concentration (CMC), they self-assemble into
micelles.
The most studied Pluronic ® P85 showed the ability to enhance the
BBB permeability of a wide range of drugs, including doxorubicin,
etoposide, taxol, 3′-azido-3′-deoxythymidine, valproic acid and
loperamide, in the bovine brain microvessel endothelia cell monolayer
[274].
Evidence suggests that the inhibition mechanisms of Pluronic ®
block copolymers on P-gp activity in the BBB involve 1) copolymer interaction with the cell membrane, producing a “membrane fluidization” effect; 2) inhibition of P-gp ATPase activity; 3) depletion of
cellular ATP [273]. Batrakova et al. demonstrated in their study
using bovine brain microvessel endothelial cells that both HLB and
PO block length are important determinants for the P-gp inhibition
activities of Pluronic ® block copolymers [275]. They conclude that lipophilic Pluronic ® with intermediate length of PO (30–60 units) and
HLB b20 are most effective at inhibiting the P-gp efflux in brain
microvessel endothelia cells.
The effect of Pluronic® P85 on facilitating drug transport across the
BBB evaluated in vitro and in vivo [276] demonstrated that coadministration of 1% Pluronic ® P85 increased digoxin penetration of
the brain by threefold in vivo. Further studies showed that Pluronic®
P85 also inhibits MRP1 and MRP2 ATPases but to a lesser extent [277].
One of the interesting studies conducted by Batrakova et al. [278]
showed that when Pluronic® P85 was used at high concentration
(>CMC), the model drug, rhodamine 123 (R123) would be solubilised
inside the micelles, enter the cell and then be recycled back with a net
decrease in R123 transport across the cell monolayer. In contrast, at a
concentration below CMC, Pluronic ® P85 increased BBB permeability
of R123 in vitro. However, after Pluronic ® P85 was conjugated to insulin, the Pluronic ® P85 micelles enhanced permeability of R123, suggesting that modified micelles might pass through brain microvessel
endothelia cells via insulin RMT [278]. This has important implications
for the use of Pluronic ® P85 in drug delivery systems.
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Y. Chen, L. Liu / Advanced Drug Delivery Reviews 64 (2012) 640–665
These studies have profound implications as they suggest that
polymers are not inert; that they may have important biological activity which could be beneficial or detrimental to a drug therapy if they
are used as pharmaceutical excipients or as drug delivery vehicles.
Because the action of Pluronic ® block copolymers involves interaction with cell membrane and direct inhibition of ATP activity, a concern was raised about the potential toxicity of the system on the BBB.
Several pieces of evidence, however, show that ATP depletion by
Pluronic ® P85 was transient and that there was no compromise of
BBB integrity after ATP depletion as demonstrated by the fact that
there was no change in permeability of mannitol (a permeability
marker) both in vitro and in vivo [274,276]. In addition to their application in enhancing drug transport into the BBB, Pluronic ® block copolymers have been used to enhance activity of anticancer drugs
such as doxorubicin due to its ability to chemosensitize MDR tumours
by inhibiting P-gp and enhancing pro-apoptotic signalling in cancer
cells [279]. A Pluronic ®-based micelle formulation of doxorubicin
(SP1049C) has now entered Phase III clinical trial and received orphan drug status from the FDA in 2008. It is possible that following
the completion of the clinical trial, this formulation can also be
adopted for transporting CNS drugs, possibly in combination with a
nanocarrier system and BBB targeting ligands.
Besides Pluronic ® block copolymers, liposomes, nanoparticles and
even macromolecular conjugates can also be used to bypass P-gp and
MDR efflux pumps. Potentially, Pluronic ® copolymers could be used
in the formulation of all these types of drug delivery systems, providing additional function as a biological response modifier [76]. No
doubt, this will be a sensitive research topic as we are moving into
areas of using polymers with certain biological activities. Caution
has to be taken. It is acceptable for the delivery of cancer treatment
but it may not be suitable for treatment of chronic diseases that require long term therapy.
6. Cell-mediated drug transport across the blood–brain barrier
Cell-mediated drug transport employs specific cells that take up
drug-loaded nano or microcarriers traffic them through the BBB and
deliver the drugs to their target sites inside the brain. In this case,
cells act as Trojan horses. The rationale for cell-mediated trafficking
of drug across the blood brain barrier is based on the following:
1) During the process of inflammation in the brain (commonly occurring in AD, Parkinson's disease, MS, stroke, brain tumours and
HIV-1 associated dementia) there is extensive recruitment and
trafficking of leukocytes (monocytes and neutrophils) in the
brain. Mononuclear phagocytes (MP) and T cells would migrate
towards the site of inflammation involving the processes known
as diapedesis and chemotaxis.
2) Cells such as macrophages and monocytes/neutrophils are phagocytic and have a tendency to endocytose colloidal materials, for
example, nano or microparticles, liposomes and subsequent exocytosis to release drug and/or colloidal materials to external
media [280–282].
3) A high payload of drug can be incorporated/loaded into nanocarriers or microcarriers, then taken up by Trojan horse cells.
4) Cell-mediated transport can be combined with magnetic particles
and application of a magnetic field to further enhance the brain
delivery and provide the diagnosis of inflammation sites and
tracking of nanomaterials.
5) Potentially block one of the routes that pathogens may take for invasion of the brain.
Compared to other transport pathways, cell-mediated drug delivery has attracted far less attention for brain drug transport but there
have been some very promising results reported. Most noteworthy
is the work carried out by Jain et al. [25] on RGD-anchored magnetic
liposomes for monocytes/neutrophils-mediated brain targeting using
an IL-1β induced brain inflammation rat model. In this study, the authors used RGD as the targeting ligand for integrin receptors
expressed on neutrophils and monocytes to facilitate cell uptake of liposomes containing the anti-inflammatory drug diclofenac. A magnetic field of 8.0 kG strength was applied near the brain of rats
receiving the treatment. The uptake of drug/liposomes by cells was
improved with RGD modification, increasing to about 16%. The cell
sorting study conducted on the blood samples collected from the animal study showed RGD improved both the monocyte and neutrophil
count, by 6% and 20% respectively. The most striking result is that by
the incorporation of magnetic particles and application of a magnetic
field, the percentage drug dose that reached the brain was elevated
from 3.25% to 21.53% for RGD-modified magnetic liposomes and
from 1.86% to 14.11% for unmodified magnetic liposomes. It is a
huge success for BBB targeting. Moreover, the RGD-modified magnetic liposomes also dramatically reduced the liver uptake of the drug
from 51.43% down to 26.58%; and the unmodified 47.32% down to
29.68%. This reduction of liver uptake is just as important as the improvement in brain targeting as it will potentially reduce the side effects in liver tissues. Furthermore, the RGD-modified magnetic
liposomes also halved the dose that usually reaches the spleen. In
terms of brain accumulation, RGD-modified magnetic liposomes was
found to be 9.1 times of the free drug and 6.6 times of nonmagnetic RGD liposomes and 1.5 times of non-RGD magnetic liposomes [25] These results indicate that the guidance of an external
magnetic field augments the active targeting of drug to the brain as
well as the recruitment of leukocytes at the site of inflammation. If
this targeting effect can be translated into drug efficacy without toxicity, then this combined strategy will revolutionize the treatment
of all CNS diseases which have an inflammatory component. The potential level of toxicity arising from repeated dosing of magnetic materials must be addressed before this approach can be developed into
a clinical treatment.
It is noteworthy that the vesicle size used in the above study was
large, at 1.2 μm. It is assumed this is to allow the incorporation of both
drug and magnetic particles. Nevertheless, this also indicates that the
cell-mediated approach does not require nano-sized carriers, a feature very different from that of all other BBB targeting approaches.
In fact, a relatively large size may be necessary to allow a sufficient
number of magnetic particles to be loaded into liposomes and later
guided to the target. However, the toxicity of these vesicles needs to
be assessed. Previously it has been reported that if the vesicle size is
greater than 1 μm this could cause adverse effects after accumulation
in the lungs, causing thrombosis [283].
Qin et al. have reported a similar RGD-liposome approach for the
delivery of ferulic acid to monocytes/neutrophils in brain [284]
using the same inflammatory animal model as described by Jain et
al. [25]. The results of the study showed that 72% of RGD-liposomes
(152 nm) associated with leukocytes (monocytes and neutrophils)
while only 19.8% for plain liposomes (155 nm). Despite the liposomes
being non-magnetic, drug-loaded RGD-liposomes reached the brain
at a level 6-fold higher than that of the drug solution and 3-folds
higher than that of the drug-loaded plain liposomes, indicating that
the strategy of targeting leukocytes for brain delivery of drug worked.
Furthermore, the drug concentration in the brain appeared to have
dropped little over the period of 120 min. However, as expected,
there was a very high level of drug in the liver and the spleen. As
the data was reported as the concentration of drug in organ (μg/g),
it is impossible to compare this data with others.
In contrast to the previous two studies, Afergan et al. used conventional negatively charged liposomes without a targeting ligand to deliver serotonin to the brain via monocytes [117]. Endocytosis of
liposome by monocytes was found to be 60%, and 28.5% by granulocytes. Four hours post i.v. injection, drug concentration in the brain
was found to be 0.138% and 0.068% of dosage for drug-loaded
Y. Chen, L. Liu / Advanced Drug Delivery Reviews 64 (2012) 640–665
liposomes and free drug solution, respectively. The majority of the
injected dose was in the spleen, liver, blood and lungs. This study
was designed elegantly using 3H-liposomes and 14C-serotonin for
co-localization imaging, which confirmed that liposomes reach the
brain intact. Furthermore, the group studied the liposome distribution following injection of liposomal aldendronate (a monocyte depletion agent) and found that the treatment with liposomal
alendronate labelled with a fluorescent probe resulted in no fluorescence in brain tissue which indirectly demonstrated that the circulating monocytes are the carriers of liposomes to the brain [117].
Nevertheless, it is noted that the group used healthy rabbits and
rats for brain penetration and body distribution studies which
would not give an opportunity for recruitment of monocytes in the
brain region. This may explain why the results obtained in this
study are so different compared to previous groups'. Indirectly it
demonstrates that brain targeting by cell-mediated transport is
most suitable for treating brain diseases under inflammation.
Research has shown that both endocytosis and exocytosis process
can be modulated. For instance, Panyam showed that the dynamics of
endocytosis and exocytosis of PLGA nanoparticles in cells can be
influenced by concentration, time and energy [281]. Endocytosis is
much faster than exocytosis and exocytosis can be almost completely
inhibited by depletion of serum in the medium [281]. In the study of
liposome uptake by human peripheral blood monocytes, Mehta et al.
reported negatively charged liposomes have a more rapid uptake
rate than positively charged liposomes (3-fold increase) and neutral
liposomes (5-fold increase). The drug is released slowly from the
cells after endocytosis [285]. This suggests that by surface modification of the nanocarrier, their uptake into and release from cells can
be modulated.
Bender et al. reported the use of nanoparticles to deliver HIV protease inhibitor saquinavir into human monocytes/macrophages [286].
An in vitro test showed that the nanoparticle formulation is more potent than the free drug. It improved the delivery of antiviral agents to
mononuclear phagocyte systems.
It is noteworthy that this field research has up to now focused
more on delivery of immunomodulators for activation of cells
[287,288], and on the delivery of magnetic particles for diagnosis purposes [289]. This approach does have potential as it can virtually
deliver any types of drugs to the brain; whether hydrophilic, hydrophobic, large, small, proteins, peptides or gene constructs. In addition,
because it targets monocytes/macrophages, it will also be effective
for delivering drugs against pathogens which infect leukocytes. As
previously shown by Jain et al. [25], the combined strategy of magnetic liposomes and monocytes targeting ligands can maximise the
monocytes' recruitment at the brain and may prove to be a very
effective approach for drug delivery to the diseased brain which has
an inflammation component. However, the safety of this route has
yet to be fully assessed. It is unknown whether its combination with
the use of magnetic particles may lead to any iron toxicity in long
term use. Furthermore, the selectivity and maximum capacity of
this strategy for delivery of drug across the BBB warrants further
study.
7. Conclusions and remarks
In the past decade, we have seen tremendous attention and effort
focused on the development of modern and novel drug delivery systems to circumvent the BBB. This is due to the significant challenge
faced by industry, government and academics in seeking effective
drug therapies for the increasing incidence of brain disease associated
with an ageing population. In this review, we looked at the barrier
issue from a biological and pathological perspective to provide a better insight to the challenges and opportunities associated with the
BBB. We need to remind ourselves that we are developing drug delivery systems which ultimately will transport a drug to a diseased
659
brain, not a healthy one. The delivery system, including the targeting
ligands should be designed based on knowledge of the diseased BBB.
Unfortunately most of the models used in the literature for assessing
the biodistribution or pharmacokinetics of drug delivery systems are
healthy mice, rats and rabbits. They could be very different to the diseased brain, therefore the data generated on healthy animals sometimes could be inaccurate or even misleading. Our first conclusion is
therefore that in vivo models that reflect the true diseases should be
adopted to evaluate a drug delivery system adequately. The urgent
need of standardised diseased animal models that are accessible to
all must be considered and met to advance the research and development in drug transport across the BBB.
To successfully target the brain, the selectivity of a BBB receptor is
extremely critical. This means, ideally, the receptor should be brain
specific, or at least, preferentially expressed at the BBB. Unfortunately
almost all the receptors are nearly non-specific as shown by percentage dose reached the brain compared to that reached by the liver,
spleen or lung. To date the best targeting in the BBB we have seen is
21% (of administrated dose) in rat and 26.6% in liver by Jain et al.
[25] achieved via cell-mediated transport using RGD-modified magnetic liposomes under magnetic guidance, which takes advantages
of immune-response occurred at the BBB under inflammatory conditions. We believe that the reason for this high level of targeting is the
design of the delivery system that is based on the pathological condition of the BBB and the fact that the system was tested on a diseased
model. The targeting effect was greatly enhanced further by its combination with the guidance of an external magnetic field. In contrast,
while RMT worked alone, the best brain targeting, as far as authors
are aware, was achieved by g7 molecule which was characterised as
a high but short lived targeting (14% at peak) [265]. Unfortunately
the majority only achieved 1–4% [290] as a result of poor selectivity
and low BBB permeability. Although the amount reaching the brain
may be sufficient for a therapeutic effect, the loss of 96–99% is huge
from the cost point of view [290], in addition to the potential sideeffects and toxicity that can be caused by 96% of drugs. Jones and
Shusta [290] suggest the solution may be in combinatory antibody library technology which allows the development of BBB-specific
RMT. Tosi et al. showed that the application of double targeting ligands can provide added targeting benefit [265]. Therefore our second conclusion is that use of multiple targeting ligands developed
with consideration of the pathological conditions of the disease will
maximise success.
Ideally the treatment for a brain disease should be constantly
monitored and modulated. The idea of using magnetic or bubblefilled and drug-loaded nanocarriers, in combination with MRI and ultrasound, may provide a solution for achieving both the diagnosis and
treatment purposes. This requires cross-discipline fertilisation of innovative ideas and multi-discipline approach to the design, characterisation and evaluation of the systems. Our third conclusion, therefore,
is to take the multidiscipline approach with innovative ideas to ultimately achieve the goal of delivering both diagnosis and drug therapy, selectively and efficiently, across the BBB.
Our final conclusion is that any brain-targeted delivery systems
must be assessed for their safety, risk and benefit for patients.
Currently the safety issue has been largely overlooked during the research stage, yet this issue will become critical when the drug to be
delivered is for a long term therapy. It is extremely important that
any delivery systems developed should have no significant impact,
short or long term, on the functions of the brain.
Acknowledgements
The authors wish to thank Dr Heather Benson for reviewing and
proofreading the manuscript; Sonya Chu for preparation of the graphic abstract; Michelle Robert-Libia for drawing Figs. 1 and 2.
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