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Vol. 54, Issue 4, 561-587, December 2002
Laboratory of Iron Metabolism, Department of Applied Biology and Chemical Technology, Hong Kong Polytechnic University, Kowloon, Hong Kong (Z.M.Q., H.L., K.H.); and Department of Chemistry, University of Hong Kong, Hong Kong (H.S.)
Abstract
I. Introduction
II. Transferrins
A. Occurrence and Biological Function
B. General Structural Features
III. The Transferrin Receptors
A. Structure of the Transferrin Receptor 1
B. Regulation of Transferrin Receptor 1 Expression
C. Association of Transferrin Receptor 1 with the Hemochromatosis Protein HFE
D. Second Transferrin-Binding Protein: Transferrin Receptor 2
IV. Transferrin Receptor-Mediated Iron Uptake
A. Transferrin-Bound Iron Uptake by Cells
B. Iron Transport Across the Blood-Brain Barrier
V. Transferrin As a Metallodrug Mediator
A. Complexation of Metal-Based Drugs with Transferrin
B. Structural Studies of the Complexes of Therapeutic Metal Ions with Transferrin
C. Cellular Uptake of Therapeutic Metal Ions via Transferrin Receptor-Mediated Endocytosis
1. Ga3+ and In3+.
2. Bi3+, Ti3+ and Ru3+.
VI. Transferrin Conjugates in Site-Specific Drug Delivery
A. General Methods of Preparation of the Conjugates
1. Chemical Linkage.
2. Protein Engineering.
B. Cellular Uptake and Efficacy of the Conjugates
1. Transferrin-Doxorubicin.
2. Transferrin-CRM107.
3. Others.
VII. Transferrin in Gene Delivery
A. Transferrin-Polylysine-DNA Conjugates
1. General Methods of Preparation.
2. Uptake of DNA Particles.
3. Problems Associated with Transferrin-Polylysine-Based Gene Delivery.
B. Transferrin-Lipoplexes
C. Other Vectors Using Transferrin as a Targeting Ligand
VIII. Transferrin and Transferrin Receptor in Drug and Gene Delivery across the Blood-Brain Barrier
A. OX26 As an Efficient Brain Drug Transport Vehicle
B. Preparation of OX26 Drug Conjugates
C. Delivery of Therapeutics to the Brain
IX. Summary
Acknowledgments
References
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Abstract |
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The membrane transferrin receptor-mediated endocytosis or internalization of the complex of transferrin bound iron and the transferrin receptor is the major route of cellular iron uptake. This efficient cellular uptake pathway has been exploited for the site-specific delivery not only of anticancer drugs and proteins, but also of therapeutic genes into proliferating malignant cells that overexpress the transferrin receptors. This is achieved either chemically by conjugation of transferrin with therapeutic drugs, proteins, or genetically by infusion of therapeutic peptides or proteins into the structure of transferrin. The resulting conjugates significantly improve the cytotoxicity and selectivity of the drugs. The coupling of DNA to transferrin via a polycation or liposome serves as a potential alternative to viral vector for gene therapy. Moreover, the OX26 monoclonal antibody against the rat transferrin receptor offers great promise in the delivery of therapeutic agents across the blood-brain barrier to the brain.
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I. Introduction |
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The rapid development in current pharmaceutical drug discovery has
resulted in the emergence of increasing numbers of novel therapeutic
drugs for the treatment of a variety of diseases. However, at present
the main problem associated with systemic drug administration is likely
to include even biodistribution of pharmaceuticals throughout the body,
the lack of drug-specific affinity toward a pathological site,
nonspecific toxicity, and other side effects resulting from high doses.
An attractive strategy to enhance the therapeutic index of drugs is to
specifically deliver these agents to the defined target cells thus
keeping them away from healthy cells, which are sensitive to the toxic
effects of the drugs. Polymer- and liposome-based delivery systems show
potentials as specific and target-oriented delivery systems (Langer,
1998
; Maruyama et al., 1999
; Vyas and Sihorkar, 2000
; Vyas et al.,
2001
). Naturally existing proteins (such as transferrin) have also
received major attention in the area of drug targeting since these
proteins are biodegradable, nontoxic, and nonimmunogenic. Moreover,
they can achieve site-specific targeting due to the high amounts of their receptors present on the cell surface. The efficient cellular uptake of transferrin
(Tf1) pathway has
shown potential in the delivery of anticancer drugs, proteins, and
therapeutic genes into primarily proliferating malignant cells that
overexpress transferrin receptors (TfRs) (Kratz and Beyer, 1998b
;
Singh, 1999
; Vyas and Sihorkar, 2000
; Kircheis et al., 2002
).
In this review, we summarize the biochemistry and molecular biology of Tf and the TfR, including the structure, function, and regulation of TfR expression as well as the latest progress in understanding the mechanism of TfR-mediated iron uptake by cells and transport across the blood-brain barrier. In particular, we address the role of Tf and TfR in the targeted delivery of therapeutic drugs, including small molecule drugs, therapeutic peptides, proteins, and even genes, into the malignant tissues or cells. The role of TfR in brain drug targeting and delivery is also included.
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II. Transferrins |
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|
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During the past decades, intensive studies have been made toward
understanding these unique iron-binding proteins. Several reviews have
given a broad range of coverage of Tf and TfR functional properties,
structures, metal binding properties, and their potential in biomedical
processes (Baker, 1994
; Morgan, 1996
; Richardson and Ponka, 1997
;
Aisen, 1998
; Sun et al., 1999
; Andrews, 2000
; Lieu et al., 2001
).
A. Occurrence and Biological Function
The transferrins comprise a family of large (molecular mass ca. 80 kDa) nonheme iron-binding glycoproteins, believed to originate with the
evolutionary emergence of vertebrates or prevertebrates. Three major
types of transferrins have been characterized. Serum Tf occurs in blood
and other mammalian fluids including bile, amniotic fluid,
cerebrospinal fluid, lymph, colostrom, and milk. Ovotransferrin (oTf)
is found in avian and reptilian oviduct secretions and in avian egg
white (Jeltsch and Chambon, 1982
; Williams et al., 1982
), and
lactoferrin (Lf) is found in milk, tear, saliva, and other secretions
(Baggiolini et al., 1970
; Metzboutigue et al., 1984
). Transferrin is
mainly synthesized by hepatocytes, with a concentration of 2.5 mg/ml
and 30% occupied with iron in blood plasma (Leibman and Aisen, 1979
).
Recently, a new member of the transferrin family, melanotransferrin
(also called p97), has been identified as an integral membrane protein
in human malignant melanoma cells and in some fetal tissues (Brown et
al., 1982
; Rose et al., 1986
). It can also be expressed by a wide range
of cultured cell types, including liver and intestinal cells (Qian and
Wang, 1998
).
The principal biological function of transferrins, except
melanotransferrin, is thought to be related to iron binding properties. Serum Tf has the role of carrying iron from the sites of intake into
the systemic circulation to the cells and tissues (Morgan, 1964
; Baker
and Morgan, 1969
). It is also likely to be involved in the
transportation of wide range of other metal ions other than iron,
including therapeutic metal ions, radio diagnostic metal ions, and some
toxic metal ions (Savigni and Morgan, 1998
; Sun et al., 1999
).
Lactoferrin, on the other hand, is believed to act principally as a
bacteriostat, by chelating the iron, which is essential for the growth
of microorganism antimicrobial activity. Lactoferrin may also be
involved in modulation of the immune and inflammatory responses and act
as a growth factor, which is unrelated to iron binding properties (Iyer
and Lönnerdal, 1993
; Lönnerdal and Iyer, 1995
).
Ovotransferrin also exhibits antimicrobial activity. The function of
melanotransferrin is not yet clear. It seems true that it plays a small
role in iron uptake. Instead, it may help in the rapid proliferation of
tumor cells and also act as an iron scavenger at the cell surface to
prevent lipid peroxidation (Kwok and Richardson, 2002
).
B. General Structural Features
The primary structures of over 10 transferrins have been
determined and can be found in various protein databases. Transferrins are single-chain glycoproteins containing ca. 700 amino acids with
molecular mass ca. 80 kDa. The sequence identity between different
species and different members of the family is extremely high, e.g.,
78% identity between rabbit and human serum transferrin, 60% between
serum transferrin and lacoferrin, and ca. 40% identity between
melanotransferrin and other transferrins. The high levels of
conservation in their primary structures were also reflected in their
three-dimensional structures. Many crystal structures of transferrins
(different species and some fragments) are available and have been
reviewed previously (Sun et al., 1999
). Briefly, the polypeptide chain
is folded into two structurally similar but functionally different
lobes, referred to as N- and C-lobe, respectively. Two lobes were
connected by a short peptide. Each lobe can be further divided into two
domains enclosing a deep hydrophilic cleft bearing an iron binding
site. At the metal binding site, Fe3+ coordinates
with distorted octahedral geometry to two oxygens from Tyr, one
nitrogen from His, one oxygen from Asp, and two oxygens from a
bidentate carbonate (synergistic anion). The ligands are from two
domains and two polypeptide strands, which cross over between the two
domains at the back of the iron site. This kind of arrangement is
crucial to ensure that the domains are able to move apart to form an
open conformation, hinged by the backbone strands, which leads to iron
release. Bicarbonate is essential for strong binding of iron to the
specific site of Tf and may also have a role in iron release. In
addition to iron, many metal ions other than iron have been found to
bind to the specific iron sites (Sun et al., 1999
; Zhong et al., 2002
),
thus Tf has been implicated in the transportation of other metal ions.
Another distinctive feature of Tf is that it undergoes conformational
changes during Fe3+ uptake and release that are
thought to be crucial for the selective recognition by the receptor of
the transporter protein. The mechanism for opening and closing the
lobes has been studied intensively but still remains elusive. It has
been postulated that the dilysine (Lys209-Lys301 for ovotransferrin)
pair may serve a special function in the release of iron. The charge
repulsion resulting from the protonation of the dilysine, located in
opposite domains, at lower pH may be the trigger to open the cleft and
facilitate iron release (Dewan et al., 1993
). In addition, this
dilysine pair may also serve as an anion binding site for iron release
(Baker, 1994
; He et al., 1999a
). Mutation of either or both lysines to
glutamate or glutamine would abolish this trigger and result in an
extremely slow release of iron compared with that from the intact
N-lobe Tf (He et al., 1999a
). For human serum Tf N-lobe, the presence of a trigger mechanism for the domain closure has been claimed on the
basis of the absence of full closure in the
Fe3+-loaded Asp63Ser mutant, as analyzed by X-ray
solution scattering (Grossmann et al., 1993a
). However, the X-ray
crystal structure of Asp60Ser lactoferrin showed a greater domain
closure than the parent half-molecule (N-lobe) lactoferrin, which has
led to the proposal of an equilibrium between the open and closed forms
in solution with a low energy barrier (Faber et al., 1996
). Recently, a
different trigger mechanism for domain closure was proposed, such that
the small triggered motion in Tyr92, an
Fe3+-coordinating ligand, would induce the
extensive rearrangements in the hydrogen bonding networks in the
-strand where Tyr92 is located. These networks would work as a
driving force for the domain closure (Mizutni et al., 2001
).
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III. The Transferrin Receptors |
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A. Structure of the Transferrin Receptor 1
The primary structure of the human transferrin receptor 1 (TfR1)
has been deduced from the nucleotide sequence of its cDNA (McClelland
et al., 1984
; Schneider et al., 1984
). It is a transmembrane homodimer
that consists of two identical monomers with a molecular mass of
approximately 90 kDa; each monomer is joined by two disulfide bonds at
Cys89 and Cys98 (Jing and Trowbridge, 1987
). It has a short,
NH2-terminal cytoplasmic region (residues 1 to
67), a single transmembrane pass (residues 68 to 88), and a large
extracellular portion (ectodomain, residues 89 to 760), which is
soluble and bears a trypsin-sensitive site and contains a binding site
for transferrin. TfR1 is synthesized in the endoplasmic reticulum and
is post-translationally modified with both phosphate and fatty acyl
groups (Omary and Trowbridge, 1981
; Schneider et al., 1982
). The
extracellular domain contains three N-linked glycosylation sites at
Asn251, Asn317, and Asn727 and one O-linked glycosylation site at
Thr104 (Omary and Trowbridge, 1981
). These sites are thought to be
crucial for the function of TfR1. Mutations at the N-linked glycosylation sites impair transferrin binding activity. Similarly, elimination of the O-linked glycosylation at Thr104 enhances the cleavage of TfR1 and promotes the release of its ectodomain (Williams and Enns, 1991
; Rutledge and Enns, 1996
).
Crystallographic studies of the ectodomain of human TfR1 (residues 122 to 760) revealed that homodimer of TfR1 is organized as a
butterfly-like shape (Fig. 1). Each TfR1
monomer consists of three distinct globular domains (Lawrence et al.,
1999
). Three domains, identified as the protease-like, apical, and
helical domains, form a lateral cleft, which is likely to be in contact with the docked transferrin molecules (Lawrence et al., 1999
). The
ectodomain of TfR1 is separated from the membrane by a stalk, which
probably includes residues involved in disulfide bond formation and the
O-linked glycosylation (Fuchs et al., 1998
). The amino acid sequence of
the globular ectodomain of TfR1 is 28% identical to that of membrane
glutamate carboxypeptidase II, which hydrolyzes the most prevalent
mammalian neuropeptide,
N-acetyl-
-L-aspartyl-L-glutamate (Lawrence et al., 1999
). Therefore, it has been suggested that TfR1 is
evolved from a peptidase related to membrane glutamate carboxypeptidase
II (Bzdega et al., 1997
) although it lacks peptidase activity.
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A homodimer of TfR1 can bind up to two molecules of Tf. The binding
affinity of Tf for various receptors is very high, from 105 to 1010
M
1 (Sun et al., 1999
). Small differences
between transferrin receptors can have large effects on the binding
affinity for Tf from different mammals and different cells. However,
the diferric protein always has a higher affinity for the TfR1 than its
mono-ferric and apo-forms. It is not fully understood how and where TfR
binds Tf. The primary receptor recognition site of human transferrin is
thought to be mainly on the C-lobe of Tf (Zak et al., 1994
). However,
recent studies show that both C- and N-lobe of human serum Tf are
necessary for receptor recognition (Mason et al., 1997
; Zak et al.,
2002
). Other studies using a human/chicken chimeric TfR1 suggest that Tf binds to a region corresponding to the helical domain (Buchegger et
al., 1996
). Site-directed mutagenesis has shown that TfR1 residues 646-648, which are present in helix 3 of the helical domain, are critical for Tf binding (Dubljevic et al., 1999
). However, docking of
Tf molecules to the crystal structure of TfR1 shows that the most
contact between Tf and TfR1 involves the apical domain of TfR1 and the
N1 and C1 domains of Tf,
although certain parts of the protease-like and helical domains may
also participate in binding of Tf (Lawrence et al., 1999
). Therefore,
further studies are warranted to understand how TfR1 binds Tf.
B. Regulation of Transferrin Receptor 1 Expression
The TfR1 appears to be expressed in all nucleated cells in the
body, such as red blood cells, erythroid cells, hepatocytes, intestinal
cells, monocytes (macrophages), brain, the blood-brain barrier (also
blood-testis and blood-placenta barriers), and also in some insects and
certain bacteria (Levay and Viljoen, 1995
; Lönnerdal and Iyer,
1995
; Schryvers et al., 1998
; Qian et al., 1998
, 1999a
, 2000
; Moos and
Morgan, 2000
), but differs in levels of expression (Davies et al.,
1981
; Enns et al., 1982
). It is expressed on rapidly dividing cells,
with 10,000 to 100,000 molecules per cell commonly found on tumor cells
or cell lines in culture (Inoue et al., 1993
). In contrast, in
nonproliferating cells, expression of TfR1 is low or frequently undetectable.
Expression of TfR1 in nonerythroid cells is regulated at the
post-transcriptional level by interactions of iron-regulatory proteins
(IRPs) and iron-responsive elements (IREs) in the 3'-untranslated region of TfR1 mRNA. The 3'-untranslated region of receptor mRNA contains a series of five hairpin stem-loop structures required for
iron-dependent regulation (Casey et al., 1988
). The stem-loop structures called IREs are recognized by trans-acting
proteins, known as IRPs (Leibold and Munro, 1988
), which control the
rate of mRNA translation or stability (Rao et al., 1986
; Müllner
and Kühn, 1988
). Two closely related IRPs (IRP1 and IRP2) have
been identified to date (Leibold and Munro, 1988
; Henderson et al., 1993
). Both display IRE binding properties under conditions of iron
deprivation. IRP1 shares 30% sequence identity with
m-aconitase, a [4Fe-4S]-containing enzyme that catalyzes
the isomerization of citrate to isocitrate. IRP1 has been regarded as a
bifunctional "sensor" of iron, switching between RNA binding and
enzymatic activities as aconitase depending on cellular iron status
(Constable et al., 1992
; Haile et al., 1992
; Basilion et al., 1994
).
Under conditions of iron deficiency, IRP1 binds to the IREs in the
5'-untranslated region of ferritin mRNA (Hentze et al., 1987
; Bhasker
et al., 1993
). This binding sterically prevents the recruitment of 43 S
translation preinitiation complex and inhibits translation of ferritin.
On the other hand, binding of IRP1 to IREs in the 3'-untranslated region of TfR1 mRNA increases mRNA stability and synthesis of TfR1
(Müllner and Kühn, 1988
; Koeller et al., 1989
;
Müllner et al., 1989
). In contrast, under high concentration of
intracellular iron, IRP1 is enzymatically active and lacks RNA binding
activity that leads to the degradation of TfR mRNA, whereas ferritin
mRNA is translated efficiently (Koeller et al., 1989
; Müllner et
al., 1989
).
IRP2 shares about 62% identity with IRP1 and, despite sequence
similarities, it does not form a [4Fe-4S] cluster and consequently lacks aconitase activity (Guo et al., 1994
). IRP2 binds specifically to
all known mRNA IREs with an affinity equally as high as that of IRP1;
however, their binding specificities to distinct sequences of
iron-responsive elements differ significantly (Henderson et al., 1993
;
Guo et al., 1994
). IRP2 differs from IRP1 in the mechanism by which
iron levels are sensed (Guo et al., 1995
; Iwai et al., 1995
). IRP2
undergoes ubiquitination and proteasomal degradation in iron-replete
cells, which is mediated by an iron-dependent oxidation mechanism
requiring a unique 73-amino acid domain containing three cysteine
residues (Iwai et al., 1995
, 1998
). IRP2 is induced following iron
starvation through renewed synthesis of stable IRP2 protein and its
inactivation by iron reflects degradation of IRP2 by a
translation-dependent mechanism (Guo et al., 1994
; Henderson and
Kühn, 1995
). It is likely that IRP1 and IRP2 perform distinct
functions, probably by acting on different target genes. However,
IRP1-deficient mice show no abnormalities in iron metabolism (Rouault
and Klausner, 1997
). Therefore, IRP2 might compensate for the function
of IRP1.
The signals other than iron levels, such as nitric oxide and oxidative
stress, can also regulate IRPs and modulate cellular iron metabolism
(Drapier et al., 1993
). It has been recently reported that the
increased IRP activity induced by nitric oxide is one of the causes for
the exercise-induced low iron status and high TfR expression (Qian et
al., 1999b
, 2001
; Xiao and Qian, 2000
; Qian, 2002
). Several review
papers have given detailed information (Hentze and Kühn, 1996
;
Aisen et al., 1999
; Lieu et al., 2001
). Briefly, nitric oxide and
H2O2 produced from
oxidative stress activate IRP1 by a cycloheximide-insensitive
post-translational mechanism (Pantopoulos and Hentze, 1995
), whereas
IRP2 activation by nitric oxide requires de novo protein synthesis
(Pantopoulos et al., 1996
). Nitric oxide regulates the binding of IRP1
by disassembling the iron-sulfur cluster or by acting as a cytoplasmic
iron chelator, which results in a loss of aconitase activity (Drapier
et al., 1993
; Gardner et al., 1995
, Ho et al., 2001
; Bouton et al.,
2002
). The activation of IRP1 by
H2O2 is dependent on
extracellular signaling events (Pantopoulos et al., 1997
; Pantopoulos
and Hentze, 1998
), suggesting that cluster disassembly alone cannot
fully account for
H2O2-induced IRP1
activation and that signaling pathways are involved.
C. Association of Transferrin Receptor 1 with the Hemochromatosis Protein HFE
The TfR binds two proteins critical for iron metabolism: Tf and
HFE, the protein mutated in hereditary hemochromatosis (Pietrangelo, 2002
; Waheed et al., 2002
). Both the primary and crystal structure of
HFE showed homology to class I major histocompatibility complex protein (Feder et al., 1996
; Lebrón et al., 1998
), that is
composed of a heavy chain associated with
2-microglobulin (
2M).
The mechanism by which HFE regulates iron uptake into the body is
unknown. However, HFE was found to coprecipitate with TfR1 in tissue
culture cells (Feder et al., 1998
). The HFE·TfR1 complex can also be
identified in human tissues such as the placenta and intestine
(Parkkila et al., 1997
; Waheed et al., 1999
; Trinder et al., 2002
).
Upon forming an association complex in the endoplasmic reticulum, HFE cotrafficks with TfR through the Golgi reticulum network to reach the
cell surface (Gross et al., 1998
; Roy et al., 1999
; Salter-Cid et al.,
1999
; Ramalingam et al., 2000
). The wild-type HFE has been shown to
negatively regulate Tf-mediated iron uptake in transfected cells
(Parkkila et al., 1997
; Gross et al., 1998
; Roy et al., 1999
; Schwake
et al., 2002
). This inhibitory effect is however attenuated in the
Cys282Tyr mutant HFE (Feder et al., 1998
; Okamoto, 2002
; Schwake et
al., 2002
), suggesting that patients with HFE mutations might have
pathological iron regulation, possibly via an altered TfR1-dependent
iron metabolic pathway.
Similar to Fe-Tf, HFE binds tightly to soluble TfR1 at the cell surface
pH 7.4 with Kd ca. 0.6 nM, with little
or no binding at the acidic intracellular vesicles pH, suggesting that
HFE dissociates from TfR1 in acidified endosomes (Lebrón et al.,
1998
). Both 2:1 and 2:2 TfR1/HFE stoichiometries have been observed
(Lebrón et al., 1998
; Bennett et al., 2000
; West et al., 2001
);
however, the stoichiometry of TfR1/HFE on the cell membrane is not
known. It has been demonstrated that membrane bound or soluble HFE
binding to cell surface TfR1 results in a reduction in the affinity of TfR1 for Fe-Tf (Feder et al., 1998
; Gross et al., 1998
). This has been
attributed to the formation of the ternary complex consisting of one
Fe-Tf and one HFE bound to a TfR1 homodimer Tf (Lebrón et al.,
1998
, 1999
). The HFE-TfR1 cocrystal structure (Fig.
2) reveals that HFE binds to the
helical domain of TfR1, and a large surface area of interaction
exists between HFE and TfR1 (Bennett et al., 2000
). Binding of HFE
induces conformational changes of TfR1. The backbone structure of the
protease and apical domains remains unchanged in contrast to the
helical domain in uncomplexed TfR1, which is displaced with respect to
the other domains when compared with the structure of complexed TfR1.
The movement alters the shape of the cleft between the helical and
protease-like domains, and thus possibly alters the ability of Tf to
bind to the TfR1 (Bennett et al., 2000
). Mutation of five TfR1 residues
at the HFE binding sites results in significant reductions in Tf
binding affinity, which also support the idea that HFE and Tf compete for overlapping binding sites on TfR1 (West et al., 2001
). However, the
lower affinity of Tf for TfR1 by HFE is not a satisfactory explanation
for the lower iron uptake since the concentration of
Fe2-Tf in serum is very high (ca. 5 µM), and
the binding of Fe2-Tf to its receptor is
saturated even in the presence of HFE. A recent study showed that
overexpression of HFE without overexpression of
2M results in a decrease in TfR1-dependent
iron uptake in transfected Chinese hamster ovary cell lines, whereas
overexpression of both HFE and
2M results in
an increase in TfR1-dependent iron uptake and increased iron levels in
the cells (Waheed et al., 2002
). There is also a hypothesis that HFE
has two mutual activities in cell, i.e., inhibition of uptake or
inhibition of release of iron, and the balance between Tf saturation
and TfR1 concentrations determined which of these functions
predominates (Townsend and Drakesmith, 2002
). Functional significance
of the association of HFE with the TfR1 and the critical regulatory
role of HFE in iron metabolism remain unveiled.
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D. Second Transferrin-Binding Protein: Transferrin Receptor 2
A new TfR-like family member, transferrin receptor 2 (TfR2), has
been cloned and sequenced (Kawabata et al., 1999
). The TfR2 gene is
located on chromosome 7q22 and gives rise to two transcripts of
approximately 2.9 and 2.5 kb in length (Kawabata et al., 1999
). Amino
acid sequence analysis reveals that, like TfR1, TfR2 is a type II
transmembrane glycoprotein that shares a 45% identity and 66%
similarity in its extracellular domain with TfR1. The
-transcript
product is primarily expressed in the livers of humans and mice
(Kawabata et al., 1999
; Fleming et al., 2000
) and the TfR2
-transcript distributed widely but is expressed at low levels (Kawabata et al., 1999
). Sequence analysis of TfR2 coding and noncoding
region reveals that TfR2 does not possess IRE (Kawabata et al., 1999
).
Therefore, it is likely that expression of TfR2 is not regulated by
IRP-mediated feedback regulatory mechanism in response to cellular iron
status. Instead, it may be regulated by a different mechanism, probably
related to the cell cycle or cellular proliferation status (Kawabata et
al., 2000
). TfR2 has a similar function to TfR1 with respect to Tf
binding and Tf-mediated iron uptake. Both TfR1 and TfR2 interact with
Tf in a pH dependent manner; apo-Tf binds to these receptors only at
acidic pH and holo-Tf binds at neutral or higher pH (Kawabata et al.,
2000
). However, the affinity of TfR2 for iron-loaded Tf is 25-fold
lower than that of TfR1 for Tf (West et al., 2000
).
The expression pattern for TfR2 is distinct from that for TfR1. It has
been shown that during liver development, TfR2 was up-regulated and
TfR1 was down-regulated; during erythrocytic differentitation of murine
erythroleukemia cells induced by dimethylsulfoxide, expression of TfR1
increased, whereas TfR2 decreased (Kawabata et al., 2001a
). Therefore,
TfR2 appears to serve unique functions involved in iron metabolism,
hepatocyte function, and erythrocytic differentiation. In addition,
high levels of TfR2 expression were also found in the erythroid cell
lines including erythroid leukemia cell line. Levels of expression of
TfR2-
mRNA were found to be significantly higher in erythroleukemia
marrow samples than in nonmaligant control marrow samples, suggesting
that TfR2-
may be a useful marker of early erythroid precursor cells
(Kawabata et al., 2001b
). The clinical significance of TfR2-
expression in leukemia cells remains to be determined. Mutations in
TfR2 have been identified as the cause of a form of hemochromatosis that is not linked to the mutation of HFE (Camaschella et al., 2000
;
Roetto et al., 2001
), which suggests that TfR2 is associated with iron
overload and offers a tool for molecular diagnosis of non-HFE related disorders.
The reason for the existence of two TfRs is still not fully understood.
The high level of TfR2 expression in the liver suggests a particular
role for this receptor, possibly TfR2 contributes substantially to the
liver's ability to capture and store iron. In addition, a possibility
has recently been suggested that TfR2 might play an important role in
modulation of hepcifin expression, thus involving the regulation of
dietary iron absorption (Nicolas et al., 2001
; Fleming and Sly, 2001
,
2002
). Deficiency of TfR2 (Camaschella et al., 2000
; Roetto et al.,
2001
; Girelli et al., 2002
) has been demonstrated to cause an
hereditary hemochromatosis-like phenopyte, implicating TfR2 as a
participant in iron homeostasis. On the other hand, the observation
that the TfR1 knockout mutation in the mouse leads to an embryonic
lethal phenotype demonstrated that TfR2 couldn't fully compensate for
the functions of TfR1 (Levy et al., 1999
).
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IV. Transferrin Receptor-Mediated Iron Uptake |
|---|
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Iron is vital for almost all living organisms. However, iron
concentrations in body tissues must be tightly regulated because excessive iron leads to tissue damage as a result of the formation of
free radicals. Acquisition of iron is a challenge in which many
proteins participate to ensure that iron uptake is sufficient and
appropriate to the needs of cells, but the total number of proteins
involved in mammalian iron metabolism is unknown (Andrews, 1999
).
Recently, several new genes have been identified, which provide
insights into the mechanism of iron absorption by the enterocytes of
the duodenal mucosa (Jiang and Qian, 2001
; Ke and Qian, 2002
).
Fe3+ in food is reduced to
Fe2+ by duodenal cytochrome b (McKie
et al., 2001
) and then absorbed into the enterocyte by an iron
transporter protein DMT1 (Fleming et al., 1997
; Gunshin et al., 1997
).
Iron subsequently crosses the enterocyte and is exported from its
basolateral surface by another iron transporter, ferroportin 1 (Donovan
et al., 2000
). In the export process the iron is reoxidized to
Fe3+ by hepaestin (Vulpe et al., 1999
) and bound
to serum Tf (Aisen, 1998
). In humans, failure to maintain appropriate
levels of iron is a feature of iron-deficiency anemia, hereditary
hemochromatosis, and even certain neurodegenerative diseases (Smith et
al., 1997
; Qian et al., 1997a
; Qian and Wang, 1998
; Andrews, 1999
; Qian
and Shen, 2001
). Abnormally high levels of iron have been found in some
regions of the brain in neurodegenerative disorders (Qian and Wang,
1998
; Aisen et al., 1999
; Qian and Ke, 2001
), although it is not clear
whether iron accumulation in the brain is an initial event that causes
neuronal death or is a consequence of the disease process. It is likely
that misregulation of iron metabolism is important in the
pathophysiology of certain neurodegenerative diseases (Qian and Wang,
1998
; Qian and Shen, 2001
; Rouault, 2001
).
A. Transferrin-Bound Iron Uptake by Cells
Cells take up iron by using a variety of mechanisms. In high
organisms, one principal pathway of cellular iron acquisition is by the
receptor-mediated uptake of transferrin-bound iron, which is one of the
best understood processes in cell biology. Several review papers have
given a detailed description (Qian and Tang, 1995
; Morgan, 1996
; Qian
et al., 1997b
; Aisen, 1998
; Andrews, 1999
, 2000
; Lieu et al., 2001
).
Figure 3 shows the current model of iron
uptake from Tf via TfR1-mediated endocytosis. Briefly, the process is
triggered by the binding of Fe2-Tf to a specific cell-surface TfR1 (Morgan and Laurell, 1963
; Morgan and Appleton, 1969
). After endocytosis via clathrin-coated pits, which bud from the
plasma membrane as membrane bound vesicles or endosomes, the Fe2-Tf·TfR1 complex is routed into the
endosomal compartment. Upon maturation and loss of the clathrin coat,
the endosome becomes competent to pump protons in a process energized
by ATPase, and the endosomal lumen is rapidly acidified to a pH of
about 5.5 (Dautry-Varsat et al., 1983
; Klausner et al., 1983
; Paterson
et al., 1984
). At this pH, the binding of iron to Tf is weakened, leading to iron release from the protein. The free
Fe3+ released to endosomes is reduced to
Fe2+ on the cis-side of the endosomal
membrane probably mediated by oxidoreductase (Núñez et al.,
1990
). Fe2+ is subsequently transported out of
the Tf cycle endosome by the divalent metal transporter DMT1, i.e.,
from the endosomal membrane to the cytosol (Fleming et al., 1998
;
Tabuchi et al., 2000
). Once in the cytosol, iron is utilized as a
cofactor for aconitase, the cytochromes, RNA reductase, and heme, or
stored as ferritin. After release of iron into the endosome, the
resultant apo-Tf·TfR1 complex is then recruited through exocytic
vesicles back to the cell surface. At extracellular physiological pH,
apo-Tf dissociates from its receptor due to its low affinity at pH 7.4, is released into the circulation, and reutilized (Morgan, 1996
, 2001
;
Qian et al., 1997b
). ATP-mediated energy is necessary for sustaining TfR-mediated endocytosis and recycling (Podbilewicz and Mellman, 1990
;
Schmid and Smythe, 1991
).
|
It has been shown that the number of receptors displayed on the cell
surface is proportional to iron uptake and that iron deficiency induces
TfR1 gene expression (Aisen, 1998
), which implies the significance of
TfRs in iron uptake. Furthermore, surface display of TfR1 is affected
by its total cellular concentration, as well as its distribution and
rate of recycling between the cell surface and cell interior. The
efficiency of TfR1 function is also influenced by other proteins,
including SFT (stimulator of iron transport) and HFE. SFT stimulates
iron uptake by both Tf and non-Tf pathways (Gutierrez et al., 1997
),
whereas HFE appears to negatively module Tf-dependent iron uptake
(Parkkila et al., 1997
; Roy et al., 1999
).
B. Iron Transport Across the Blood-Brain Barrier
To date, the mechanisms of iron transport across the blood-brain
barrier (BBB) have not been completely clarified. The accumulated evidence suggests that the Tf and TfR pathway may be the major route of
iron transport across the luminal membrane of the capillary endothelium
(Bradbury, 1997
; Moos and Morgan, 1998
, 2000
; Malecki et al., 1999
),
and that iron, possibly in the form of ferrous iron, crosses the
abluminal membrane and enters into the brain, although the molecular
events of this process are unknown (Bradbury, 1997
; Moos and Morgan,
1998
). The evidence shows that the uptake of Tf-bound iron by
TfR-mediated endocytosis from the blood into the cerebral endothelial
cells is no different in nature from the uptake into other cell types
(Bradbury, 1997
). As found in other cells, this process also includes
several steps: binding, endocytosis, acidification and dissociation,
and translocation of iron across the endosomal membrane. Most of the Tf
will then return to the luminal membrane with TfR, whereas the iron
then crosses the abluminal membrane by an undetermined mechanism
(Bradbury, 1997
; Moos and Morgan, 1998
). As mentioned before, recent
studies have shown that ferroportin 1/hephaestin and/or
hephaestin-independent iron export systems might play a key role in
ferrous iron transport across basal membrane of enterocytes in the gut
(Vulpe et al., 1999
; Donovan et al., 2000
; Kaplan and Kushner, 2000
).
This process is very similar to what occurred in the BBB cells
(capillary endothelium) (Qian and Shen, 2001
). Because the form of iron
transport across this membrane might be ferrous iron (Bradbury, 1997
;
Moos and Morgan, 1998
), therefore, a ferroxidase such as hephaestin (or ceruloplasmin) might be necessary for ferrous iron to be oxidized to
ferric iron, so that iron, after crossing the basolateral membrane of
the BBB cells, could be carried away by Tf (Ke and Qian, 2001
). Based
on the similarity of the transport form of iron across the basolateral
membranes (both are ferrous iron), and the existence of ferroportin 1 and hephaestin in the brain (Jiang et al., 2002
), it is possible that
ferroportin 1/hephaestin or ferroportin 1/ceruloplasmin system might
play a role in iron transport across the BBB cells. Another proposed
mechanism involved in ferrous iron transport across abluminal membrane
is the role of astrocytes. The astrocytes probably have the ability to
take up ferrous iron from endothelial cells through their end feet
processes on the capillary endothelia (Malecki et al., 1999
; Oshiro et
al., 2000
). In addition to Tf/TfR pathway, it has been suggested that
the lactoferrin receptor/lactoferrin and GPI-anchored p97/secreted p97
pathways might play a role in iron transport across the BBB (Faucheux
et al., 1995
; Qian and Wang, 1998
; Malecki et al., 1999
). It is also
possible that a small amount of iron might cross the BBB in the form of
intact Tf·Fe complex by receptor-mediated transcytosis (Moos and
Morgan, 1998
) (Fig. 4). After the iron
has been transported across the BBB, it is likely to bind quickly to
the Tf that is secreted from the oligodendrocytes and choroid plexus
epithelial cells (Bradbury, 1997
; Moos and Morgan, 1998
) or other
transporters and then transported to where iron is needed (Qian and
Shen, 2001
).
|
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V. Transferrin As a Metallodrug Mediator |
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A. Complexation of Metal-Based Drugs with Transferrin
The transferrins are primarily iron-binding proteins, but in human
serum, Tf is only about 30% saturated with iron, so there is a
potential capacity for binding to other metal ions that enter the body.
Indeed, over 30 metal ions have been reported to bind to Tf with either
carbonate, oxalate, or other carboxylates as synergistic anions,
although Fe3+ has a higher affinity than any
other metal ion for which the binding constant has been determined
(Aisen, 1998
; Sun et al., 1999
). Such binding may play an important
role in the transport and delivery of medical diagnostic radioisotopes
such as 67Ga3+ and
111In3+ (Harris and
Pecoraro, 1983
; Ward and Taylor, 1988
; Harris et al., 1994
; Bernstein,
1998
) and therapeutic metal ions such as Bi3+ (Li
et al., 1996a
; Sun et al., 2001
; Zhang et al., 2001
),
Ru3+ (Kratz et al., 1994
; Smith et al., 1996
) and
Ti4+ (Sun et al., 1998a
; Messori et al., 1999
).
Electronic absorption spectroscopy is frequently used to detect metal
binding to the specific iron sites of transferrins. Apo-transferrin
(apo-Tf) is a colorless protein with an intense ultraviolet absorption
near 280 nm with
278 93,000 M
1 cm
1, attributable to
-
* transitions of the aromatic amino acids tyrosine, tryptophan,
and phenylalanine. The binding of metal ions to the phenolic groups of
the tyrosine residues in the specific metal binding sites of apo-Tf
leads to the production of two new absorption bands centered at ca. 240 nm and ca. 295 nm in the UV-difference spectra. This has been widely
exploited for metal titration and thermodynamic studies. Typical
difference spectra are shown in Fig. 5,
in which two new bands centered at 241 and 295 nm appear and increase
in intensity with time after addition of 2 mol Eq of
Bi3+ as [Bi(Hcit)], indicative of a slow
complexation of Bi3+ in the specific binding
sites of apo-Tf. When transition metal ions bind to apo-Tf, there are
often additional intense tyrosinate-to-metal charge transfer (LMCT)
bands in the visible region of the spectrum (400-500 nm;
ca.
4-9 × 103 M
1
cm
1) that are also diagnostic of site-specific
binding. For example, the Fe3+ complex is
orange-red with a band at ca. 465 nm, the Cu2+
and Co3+ complexes are yellow, and the
Mn3+ complex is brown (Aisen et al., 1969
).
Electronic absorption spectroscopy also allows determination of the
strength of metal binding to Tf (Sun et al., 1999
). The strength of
binding of metal ions has been found to correlate well with metal ion
acidity, and the most readily hydrolyzed (most acidic) metal ions bind most strongly to transferrin (Li et al., 1996b
; Sun et al., 1997a
). This provides a basis for the prediction of unknown stability constants
for metal·Tf complexes and allows the discovery of new metal ion
(e.g., Ti4+) binding to Tf (Sun et al., 1998a
).
Table 1 lists the binding constants for
therapeutic metal ions with Tf.
|
|
B. Structural Studies of the Complexes of Therapeutic Metal Ions with Transferrin
Iron binding to Tf induces protein structure changes from an open
to a closed conformation, which is thought to be crucial for receptor
recognition. Therefore it is important to study the structural changes
induced by other metal ions. Although many crystal structures are
available for either apo-Tf or holo-Tf, there appears very few for
other metal-Tf (Shongwe et al., 1992
; Smith et al., 1996
). X-Ray
crystallographic studies of human lactoferrin have demonstrated that
Ru3+ coordinates directly to the imidazole
nitrogen of His253, one of the Fe3+ ligands in
the iron binding cleft of the N-lobe, with displacement of a chloride
ligand. At least one indazole ligand remains coordinated to the
Ru3+ (Smith et al., 1996
). However, this study
could not provide whether the protein would form the "closed"
structure with Ru complexes. Modeling by superposition of normal closed
structure suggests that certain Ru3+ complexes
may allow closure of the protein (Smith et al., 1996
). Small angle
X-ray scattering has also been used to provide direct structural
information on the conformational changes induced by metal ions. It was
shown that In3+ induced the same domain closure
as Fe3+ (Grossmann et al., 1993b
).
Isotopic labeling of Tf can provide assignments of resonances for
specific amino acid residues of Tf, which enhances the usefulness of
NMR spectroscopy in exploring conformational changes in the protein. By
means of two-dimensional NMR and single site mutations, the
S-13CH3 resonances of all
the five Met residues of the N-lobe and nine Met residues of intact Tf
have been tentatively assigned (Beatty et al., 1996
; He et al., 1999b
).
The Met residues are well spread throughout the protein and occupy
several environments. Some of the Met resonances (e.g., Met464 and
Met109) are sensitive to metal binding even though these residues are
far away from the metal binding site (>10 Å). Similar changes in
1H/13C shifts of Met
resonances are observed for Fe3+,
Ga3+, and Bi3+ indicating
that they induce similar conformational changes (Sun et al., 1998b
).
Ti4+ also induces a similar structural change as
Fe3+ (Guo et al., 2000
). This approach provides a
useful technique for probe protein conformational changes induced by
metals under biologically relevant conditions (Sun et al., 2001
).
C. Cellular Uptake of Therapeutic Metal Ions via Transferrin Receptor-Mediated Endocytosis
There is increasing interest in the use of metal-containing
compounds in medicine, such as the use of platinum complexes in cancer
chemotherapy, gold compounds in the treatment of arthritis, gallium and
indium in diagnosis and radiotherapy, and bismuth in anti-ulcer
medication (Abrams and Murrer, 1993
). One concern is how we can ensure
that a metal-based therapeutic or diagnostic agent reaches its target
site. One way is to incorporate features that are recognized
specifically by the target. We can seek to use natural recognition
mechanisms such as the Tf/TfR recognition system. The natural Tf cycle
for the delivery of Fe3+ to cells offers an
attractive system for strategies of drug delivery and targeting since
TfR can bind strongly to a range of other metal ions apart from
Fe3+, and many heterometal·Tf complexes are
still recognized by the TfR.
1. Ga3+ and In3+.
The mechanisms by
which Ga3+ is transported into target sites are
of fundamental importance since gallium compounds have been used
extensively both in the diagnosis and the treatment of human cancers
(Tsuchiya et al., 1992
; Bernstein, 1998
). It has been shown previously
that Ga3+ binds to Tf in the specific
Fe3+ binding sites with a similar affinity,
attributed to the similarity between these two metal ions (Harris and
Pecoraro, 1983
). In vivo studies using 67Ga find
that all gallium in blood is present in plasma (with traces in
leukocytes) and is tightly bound to Tf (Clausen et al., 1974
). There
has been a quite controversy about whether 67Ga
uptake is a Tf-independent or -dependent process and whether tumors and
normal tissues differ in the mechanism of uptake. Early studies have
shown that the uptake of 67Ga by cultured murine
tumor cells can be significantly stimulated by the addition of
exogenous transferrin to the tissue culture medium (Harris and Sephton,
1977
). In contrast, a decrease in 67Ga uptake by
certain tumor cells has been noticed following the addition of Tf to
the incubation medium (Vallabhajosula et al., 1981
). The uptake study
of 67Ga3+ by human leukemic
cell line HL60 demonstrated that both a transferrin receptor-dependent
and a Tf-independent mechanism exist. HL60 cells incorporated about 1%
of the Ga3+ dose over 6 h in the absence of
Tf. However, the presence of Tf could increase cellular
Ga3+ uptake approximately 10-fold (Chitambar and
Zivkovic, 1987
). Anti-Tf receptor monoclonal antibody inhibited
Ga3+ uptake, and decrease in the density of
cellular TfR led to corresponding decreases in the Tf-dependent uptake
of Ga3+ (Chitambar and Zivkovic, 1987
). Cell
surface-bound Ga2-Tf displayed similar kinetics
as Fe2-Tf during the first 10-min uptake,
suggesting that the initial internalization of
Ga2-Tf closely resembled that of
Fe2-Tf (Chitambar and Zivkovic-Gilgenbach, 1990
).
However, unlike 59Fe, a small fraction of
internalized 67Ga was released from cells by an
unknown reason. Ammonium chloride inhibited the internalization of both
67Ga and 59Fe, indicating
that 67Ga-Tf uptake by HL60 cells involves
initial internalization into acidic receptosome and followed by
dissociation of 67Ga and Tf and subsequent
trafficking of each to separate compartments (Chitambar and
Zivkovic-Gilgenbach, 1990
). Ga2-Tf disrupts
TfR-mediated cellular uptake of iron as results of inhibition of the
iron-containing M2 subunit of ribonucleotide reductase (Chitambar et
al., 1988
, 1991
). Transferrin-enhanced uptake of
67Ga was also found in other cell lines (Table
1). Therefore, it can be concluded that the TfR pathway is of primary
importance in the incorporation of Ga3+ into the
cytoplasma of cells displaying this receptor. However, 67Ga can also enter tumors and other cells by a
Tf-independent mechanism, which is probably also used by iron
(Chitambar and Zivkovic, 1987
; Weiner et al., 1996
); this becomes
apparent when Tf is in short supply or saturated with iron or other
metal ions (Sohn et al., 1993
).
2. Bi3+, Ti3+ and
Ru3+.
Bi3+ complexes are in
widespread use in the treatment of ulcers (Sun et al., 1997b
; Sun and
Sadler, 1998
; Briand and Burford, 1999
; Sadler et al., 1999
);
Ru3+ compounds are potential anticancer agents,
which are often active against metastases but not against the primary
tumors (Kratz et al., 1994
; Clarke et al., 1999
);
Ti4+ complexes have been shown to exhibit high
antitumor activities against a wide range of murine and human tumors
with less toxic side effects than cisplatin (Köpf-Maier and
Köpf, 1987
; Harding and Mokdsi, 2000
). There are two titanium
complexes, titanocene dichloride and budotitane, now in clinical trials
(Köpf-Maier and Köpf, 1987
; Keppler et al., 1991
). All
these metal ions have been found binding to Tf strongly in the specific
iron sites (Table 1). Therefore, Tf may act as a carrier to deliver
these therapeutic metal ions into the cells. Cell uptake experiments
showed that Ti2-Tf and
Bi2-Tf can block both membrane binding and
cellular uptake of Fe2-Tf (Guo et al., 2000
).
These experiments provide evidence that both bismuth and titanium are
likely transported via a similar mechanism as iron, i.e., TfR-mediated
endocytosis. Recognition of Bi2-lactoferrin by
IEC-6 rat intestinal cells (Zhang et al., 2001
) may also have
implications for bismuth antimicrobial action. It is likely that
bismuth may block the pathway of iron transport into the bacteria and
cuts iron supply required by the bacteria for its growth. A recent
study showed that Ti4+ does not bind strongly to
DNA bases at physiological pH but forms strong complexes with
nucleotides only at low pH values (below 5) (Guo and Sadler, 2000
).
Therefore, Tf may serve as a carrier to deliver titanium complexes to
tumor cells and to prevent hydrolysis of Ti4+
complexes at neutral pH. Titanium is subsequently released as a result
of acidic microenvironment in tumors than in normal tissue (Yamagata
and Tannock, 1996
), and targets DNA. Ru3+
complexes were reported bound to both albumin and Tf with an 80%
portion binding to albumin and the remainder to the latter (Messori et
al., 2000
; Frasca et al., 2001
). Injection of
Ru3+-Tf resulted in high tumor uptake of the
metal (Som et al., 1983
; Ando et al., 1988
; Srivastava et al., 1989
),
which suggests that Tf uptake appears to be the more important mode of
transport of Ru3+ anticancer complexes to the
tumor. The Ru2-Tf exhibits a significantly higher
antitumor activity against human colon cancer cells than the
albumin-bound complex or the Ru3+ complex itself
(Kratz et al., 1994
, 1996
), probably attributed to the Tf-mediated
uptake mechanism, which may lower ruthenium toxicity by preventing it
from other binding or uptake until it has been delivered to the cells.
| |
VI. Transferrin Conjugates in Site-Specific Drug Delivery |
|---|
|
|
|---|
A. General Methods of Preparation of the Conjugates
1. Chemical Linkage.
Various therapeutic agents have been
chemically linked to Tf. Several studies have been reported to link
doxorubicin with Tf via the formation of a Schiff base (Yeh and Faulk,
1984
; Barabas et al., 1992
; Sizensky et al., 1992
; Bërczi et al.,
1993a
,b
; Singh et al., 1998
). Glutaraldehyde was frequently used for
this purpose. Briefly, certain amounts of Tf and doxorubicin both
dissolved in 150 mM NaCl were directly mixed and followed by the
addition of glutaraldehyde (in 150 mM NaCl) dropwise. The coupling
procedure was stopped by the addition of ethanolamine. The conjugate
was subjected subsequently to purification and characterization. The conjugates prepared in such a way were found to exert cytotoxicity (Yeh
and Faulk, 1984
; Barabas et al., 1992
; Bërczi et al., 1993a
,b
).
; Singh, 1999
). A new coupling approach
has been attempted, in which the stability of the bond between the Tf
and the drug can be finely tuned (Kratz et al., 1998a
). This was
achieved by synthesis of the first derivatizing the drug with a spacer
group, such as maleimide spacer, and then attaching the drug derivative
to the carrier protein (e.g., Tf). In this way, the bond between the
drug and the spacer can act as a cleavage site, allowing the drug to be released inside the cells.
2. Protein Engineering.
A novel alternative approach to using
the Tf uptake pathway for cellular delivery of therapeutic agents is to
incorporate the drug into the structure of Tf using recombinant protein
engineering (Ali et al., 1999a
,b
). A therapeutic peptide sequence
cleavable by the human immunodeficiency virus type 1 protease has been
inserted into various regions of human serum Tf by protein engineering techniques. These insertions were cloned and expressed using a baculovirus expression vector system. The results showed that mutant
proteins retained the native Tf function and that the inserted peptide
sequence was surface-exposed. Most importantly, two of the mutants
could be cleaved by human immunodeficiency virus-1 protease (Ali et
al., 1999a
,b
). In another study, Tf was fused to mouse-human chimeric
IgG3 at different positions by protein engineering, and the resulting
fusion protein was found to be able to cross the BBB and to target the
brain (Shin et al., 1995
). These studies have demonstrated the
potential of Tf not only as a carrier protein for site-specific drug
delivery, but also for developing new therapeutic agents for a broad
spectrum of diseases in the future.
B. Cellular Uptake and Efficacy of the Conjugates
1. Transferrin-Doxorubicin.
Although doxorubicin (Adriamycin)
is an effective and widely used cancer chemotheraputic agent;
cardiotoxicity and emergence of resistance tumor cell lines
significantly limit its utility in clinical practice (Kovar et al.,
2002
). Various approaches have been devised to circumvent these
limitations, among which is the attachment of cytotoxic drugs to
suitable carrier proteins, such as Tf, that accumulate in tumor tissue.
The Tf-doxorubicin conjugate has been shown to exhibit greatly
increased cytotoxicity relative to unconjugated doxorubicin toward a
variety of culture cell lines (Table 2).
Cellular uptake experiments revealed that free doxorubicin at
concentrations below 1 × 10
7 M had little
effect on K-562 cell, while Tf-doxorubicin conjugate inhibited 75% of
cellular activity. When normal peripheral blood mononuclear cells were
tested against the conjugate, the 50% inhibitory concentration was
found to be 1.4 to 1.7 × 10
6 M, at which
concentration over 85% of K-562 cells were inhibited (Sizensky et al.,
1992
). In another cytotoxicity assays, K-562 cells were exposed to
doxorubicin or Tf-doxorubicin and cultured for 16 to 18 h. It was
found that at a concentration of 0.05 µM, 37% inhibition of K-562
observed for the conjugate compared with 5% for the free drug
(Bërczi et al., 1993b
). In vivo studies showed that the life span
of tumor-bearing mice was significantly increased when they were
treated with Tf-doxorubicin conjugate (increase in life span 69%
versus 39% with doxorubicin); although no long-term survivors was
observed, the tumor burden with conjugate-treated mice was much smaller
compared with free doxorubicin-treated mice (Singh et al., 1998
).
TABLE 2
Conjugates of therapeutic agents with Tf