Endogenous carriers and ligands in non-immunogenic site-specific drug delivery

Transferrin/Transferrin Receptor-Mediated Drug Delivery

Hongyan Li, Zhong Ming Qian

Laboratory of Iron Metabolism, Department of Applied Biology and Chemical Technology,

The Hong Kong Polytechnic University, Kowloon, Hong Kong

!

Abstract: Since transferrin was discovered more than half a century ago, a considerable effort has

been made towards understanding tranferrin-mediated iron uptake. However, it was not until

recently with the identification and characterization of several new genes related to iron homeo-

stasis, such as the hemochromatosis protein HFE and the iron transporter DMT1, that our

knowledge has been advanced dramatically. A major pathway for cellular iron uptake is through

internalization of the complex of iron-bound transferrin and the transferrin receptor, which is

negatively modulated by HFE, a protein related to hereditary hemochromatosis. Iron is released

from transferrin as the result of the acidic pH in endosome and then is transported to the cytosol by

DMT1. The iron is then utilized as a cofactor by heme and ribonucleotide reductase or stored in

ferritin. Apart from iron, many other metal ions of therapeutic and diagnostic interests can also

bind to transferrin at the iron sites and their transferrin complexes can be recognized by many

cells. Therefore, transferrin has been thought as a ‘‘delivery system’’ for many beneficial and

harmful metal ions into the cells. Transferrin has also be widely applied as a targeting ligand in

the active targeting of anticancer agents, proteins, and genes to primary proliferating malignant

cells that overexpress transferrin receptors. This is achieved by conjugation of transferrin with

drugs, proteins, hybride systems with marcomolecules and as liposomal-coated systems. Con-

jugates of anticancer drugs with transferrin can significantly improve the selectivity and toxicity

and overcome drug resistance, thereby leading to a better treatment. The coupling of DNA to

transferrin via a polycation such as polylysine or via cationic liposomes can target and transfer

of the extrogenous DNA particularly into proliferating cells through receptor-mediated endo-

cytosis. These kinds of non-viral vectors are potential alternatives to viral vectors for gene therapy,

if the transfection efficiency can be improved. Moreover, transferrin receptors have shown

potentials in delivery of therapeutic drugs or genes into the brain across blood–brain barrier.

� 2002 Wiley Periodicals, Inc. Med Res Rev, 22, No. 3, 225–250, 2002; Published online in Wiley InterScience

(www.interscience.wiley.com). DOI 10.1002/med.10008

Key words: transferrin; transferrin receptor; drug delivery; gene delivery

225

Correspondence to: Zhong Ming Qian, MD, PhD, Department of Applied Biology and Chemical Technology,The Hong Kong

Polytechnic University,Kowloon,Hong Kong.E-mail: [email protected]

Contract grant sponsor: The Hong Kong ResearchGrants Council;Contract grant number: A/c:BQ-445;Contract grant

sponsor: TheHong Kong Polytechnic University Grants;Contract grant numbers: A/c:G.12.XX.93A2 andG-YW47

Medicinal Research Reviews, Vol. 22, No. 3, 225^250, 2002

� 2002 Wiley Periodicals, Inc.

1 . I N T R O D U C T I O N

An attractive strategy to enhance the therapeutic index of drugs is to specifically deliver these

agents to the defined target cells, thereby, keeping them away from healthy cells, which are sensitive

to the toxic effects of the drugs. This would allow for more effective treatment achieved with a better

tolerance. Many attempts are being made to explore the potentials of specific and target-oriented

delivery systems. Examples include polymer-based drug delivery systems, liposome-based delivery

systems, and ligand-receptor-mediated delivery systems.1–4 The latter has received major attention

in the past few years due to the potential of non-immunogenic, site-specific targeting to ligand-

specific biosites of the naturally existing ligands and their receptors. The best-characterized and

efficient cellular mechanism of uptake of transferrin (Tf) has been exploited for the delivery of

anticancer drugs, proteins, and therapeutic genes into primarily proliferating malignant cells that

overexpress transferrin receptors (TfRs).4–7 In this review, we focus on the structures of transferrin

and transferrin receptor, the regulation of transferrin receptor expression. In particular, we cover

the latest progress in understanding the mechanism of transferrin-receptor-mediated iron uptake,

the role of transferrin in delivery of therapeutic and diagnostic metal ions, and the potentials

of transferrin conjugates in drug and gene delivery. We also summarize the significance of trans-

ferrin receptors in drug and gene delivery in the brain.

2 . T R A N S F E R R I N

A. Properties and Biological Function

The transferrins are a family of iron-binding proteins, which have been the subject of intense

investigation since serum transferrin was discovered more than 40 years ago. A considerable

number of reviews have given a wide range of coverage of their functional properties, structures,

metal binding properties, and metal delivery potentials in biomedical processes.8–11

The transferrins are typically monermeric glycoproteins with a single polypeptide chain of

670–700 amino acids and a molecular weight of ca. 80 kDa. The best known members of the

transferrin family are serum transferrin found in blood and other mammalian fluids including

bile, amniotic fluid, cerebrospinal fluid, lymph, colostrom, and milk;12 ovotransferrin found in egg

white;13,14 lactoferrin found in mammalian milks and other secretions such as tears, saliva, mucus,

and white blood cells;15,16 and melanotransferrin (also called p97) found anchored to the membrane

surface of melanocytes and other cells.17 The transferrins except lactoferrin are acidic proteins

and the differic species have isoelectric poin (pI) values around 5.6–5.8; while for lactoferrin, the

pI is 8.7, which probably accounts for its ability to bind to other proteins and to cell surfaces.9

In human serum, the concentration of transferrin is about 2.5 mg/ml (35 mM) with 30% occupied

with iron.18

The fundamental role of transferrins is to control the levels of free iron in body fluids by

binding, sequestering, and transporting Fe3þ ions, which will help to maintain the availability of

iron and prevent the deposition of insoluble ferric hydroxide aggregates. Serum transferrin has the

role of transporting iron amongst the sites of absorption, storage, and utilization. It may regulate

iron metabolism and protect against the toxic side effects of free iron, which may damage cells by

catalyzing the formation of free radicals. It is likely to be involved in transportation of a wide range

of metal ions other than iron, such as therapeutic metal ions, radio diagnostic metal ions, and some

toxic metal ions.11 Ovotransferrin and lactoferrin may also have antimicrobial activity, which

apparently depends on actual contact with the bacteria rather than simple iron deprivation.19

Lactoferrin has been implicated as a growth factor and may also play an important role in

modulating the immune and inflammatory responses and in iron absorption. Detailed information

226 * LI AND QIAN

can be found in recent reviews.20,21 The function of melanotransferrin has not yet been fully

established. It may help the rapid proliferation of tumor cells, perhaps through binding and trans-

locating of iron.

B. Structure

The primary structures of more than ten transferrins have been determined.22–25 They show a high

degree of similarity with approximately 70% identity between lactoferrin, 50–60% between lacto-

ferrins and transferrins and ca. 40% between melanotransferrin and human transferrin or lactoferrin.

In each protein, the N-terminal half of the polypeptide is homologous with the C-terminal half, with

typically ca. 40% sequence identity, which is also reflected in the three-dimensional structures of the

proteins.

Crystal structures of transferrins, both intact transferrins and a number of fragments, have been

determined after the appearance of the first crystal structure of transferrin-human lactoferrin.26

These include monoferric human serum transferrin,27 diferric rabbit serum transferrin,28 diferric

bovine lactoferrin,29 diferric hen ovotransferrin,30 etc., which have been summarized in a recent

review.11 The X-ray crystal structures show that the polypeptide folding is very similar for all

proteins of the transferrin family. Transferrin is a single-chain glycoprotein with two similar lobes,

called the N- and C-lobe, connected by a short peptide (Fig. 1A). Each lobe can be further divided

into two domains of similar size, which have alternating a-helical and b-sheet segments. In each

lobe, there is a Fe3+ binding site situated in the cleft between two domains. The two iron-binding

sites are extremely similar. Fe3þ coordinated with distorted octahedral geometry to two oxygens

from two tyrosines, one nitrogen from a histidine, one oxygen from an aspartate, and two oxygens

from a bidentate synergistic anion-carbonate (Fig. 1B). The ligands are from domain 1 (Asp63),

domain 2 (Tyr188), and two polypeptide strands (Try95 and His249) which cross over between the

two domains at the back of the Fe3þ site.27 Therefore, the domains can move apart to form an open

conformation, hinged by the backbone strands, leading to iron release. Apart from Fe(III), many

divalent and trivalent metal ions have also been found bound to the specific Fe3þ sites,31 which has

led to an idea that transferrin acts as a ‘‘delivery system’’ for both harmful and beneficial metal ions

in the body.

As shown in Figure 1A, transferrin undergoes conformational changes during Fe3þ uptake and

release, which has been thought to be crucial for cell receptor recognition. The mechanism for

opening and closing lobes may involve a pH-sensitive interdomain interaction. Uptake of Fe3þ -Tf

complex into a acidic endosome (pH ca. 5.5) could result in the protonation of both residues Lys209

and Lys301 located on opposite domains, the so-called dilysine ‘‘trigger’’, which may provide the

driving force to push the two domains apart and expose the Fe3þ and facilitate its release.32 The

Asp63, an iron binding ligand, may also serve as a trigger for the closure of the two domains upon

Fe3þ uptake, since this trigger is abolished completely by the mutation of Asp63 to Ser or Cys,

which means that the lobe remains in an open conformation.33 However, a different study on

Asp60Ser lactoferrin by X-ray crystallography showed that the N-lobe is completely closed, which

has led to the proposal of an equilibrium between open and closed forms in solution with a low

energy barrier.34

3 . T R A N S F E R R I N R E C E P T O R

The transferrin receptor (TfR) assists iron uptake into vertebrate cells through a cycle of endo-

and exocytosis of transferrin (Tf).35 It appeared to be expressed in all nucleated cells in the body.

The TfR has been found in red blood cells, throid cells, heaptocytes, intestinal cells, monocytes,

brain, the blood–brain barrier, and also some insects and certain bacteria.20,36 In malignant cells,

Tf/TfR-MEDIATED DRUG DELIVERY * 227

there are elevated levels of TfR expression attributed to the requirement of high level of iron for

their growth.37 The TfR is localized on the endothelia surfaces of brain capillaries that comprise

the blood–brain barrier.38 Generally, transferrin receptors have a higher affinity to diferric trans-

ferrin than apotransferrin, and different transferrin receptors may have very different affinities to

transferrin.11

Figure 1. A: X-ray crystal structure of human serum transferrin.27The C-lobe, which contains Fe

3þbound, is shown in a closed

form (blue) and the apo N-lobe is in an open form (green). B: The metal binding sites of human serum transferrin with residue

numbersof theN-lobe inbrackets.

Figure 2. X-ray crystal structure of the dimeric ectodomain of the human transferrin receptor. It contains of three distinct domains

organized in a butterfly-like shape. The protease-like, apical, and helical domains in one monomer are shown in red, green, and

yellow, respectively; and in the otherare in blue.The transmembrane segment is shown in black and the stalk in gray connected to

theputativemembrane-spanninghelics. (Reprintedwithpermission fromLawrenceCM, etal.Crystal structure of the ectodomainof

humantransferrin receptor. Science1999;286:779^782.� American Association for the Advancementof Science.42)

228 * LI AND QIAN

A. Structure

Human TfR is a transmembrane glycoprotein composed of two disulfide-bonded (formed at Cys89

and Cys98) subunits, each of apparent molecular mass of 90 kDa.39,40 It contains three N-linked

glycan units and is post-translationally modified with both phosphate and fatty acyl groups.39,41

Each TfR monomer binds one molecule of transferrin. It has a short, N2H-terminal cytoplasmic

region (residues 1–67) containing the internalization motif Tyr-Thr-Arg-Phe, a single transmem-

brane pass (residues 68–88), and a large extracellular portion (ectodomain, residues 89–760), which

is soluble and bears a trypsin-sensitive site and contains a binding site for transferrin. Recently,

the 3D structure of the ectodomain of human transferrin receptor (residues 122–760) expressed

in Chinese hamster ovary cells has been determined at 3.2 A.42 The TfR monomer contains

three distinct domains, organized so that the TfR dimer has a butterfly-like shape in Figure 2. The

positions of the N2H-termini allow orientation of TfR with respect to the plasma membrane.

The protease-like domain consists of residues 122–188 and residues 384–606. It has a central

seven-stranded mixed b-sheet with flanking a helices and is closely related to carboxy- or amino-

peptidases.42 The apical domain contains residues 189–383. It has a b-sandwich in which the two

sheets are splayed apart with a helix running along the open edge and is related to the domain 4 of

aconitase.42 The helical domain contains residues 607–760. A four-helix bundle is formed by a pair

of parallel a-hairpins in this domain. The large loop-like insert between the 4th and 5th a -helix

may contact the apical domain and the protease-like domain and appears to play an important role

in TfR dimerization.42

It is still not fully understood how and where TfR binds Tf although a considerable effort has

been made. It has been thought that the primary receptor recognition site of human transferrin is

mainly on the C-lobe of transferrin.43 However, this has been challenged by recent studies, which

show that both C- and N-lobe of human serum transferrin are necessary for receptor recognition.44

With emergence of TfR structure, the interaction of Tf and TfR has been modeled on the basis of the

structural characteristics of the two proteins and the available functional information.42 The model

for binding of Tf to TfR is shown in Figure 3. In this model, the most contact between Tf and TfR

involves the C1 domain with additional contribution from the N1 domain. The N2 and C2 domains

of Tf have only minor interaction with TfR. The largest continuous patch of conserved surface

residues in human and rabbit Tf is at the interface between the apical domain of TfR 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.42 It could be explained with this model why the receptor enhances

iron dissociation. Presumably, TfR has conformational changes associated with pH, similar to Tf.

The motion of the apical domain transduces pH changes into changes in the Tf binding cleft which

could affect the relative affinity of TfR for apo- and holo-Tf and thus the affinity of Tf for Fe3þ .

Other studies using a human/chicken chimaeric TfR suggest that Tf binds to a region corres-

ponding to the helical domain.45 Site-directed mutagenesis has shown that TfR residues 646–648,

which are present in helix 3 of the helical domain, are critical for Tf binding.46 This is also supported

by a recent crystal structure of TfR complexed with the hereditary haemochromatosis protein HFE,

which showed that HFE binds to the helical domain of TfR.47 It is likely that Tf and HFE may bind

to the same or overlapping sites on TfRs, since competitive studies demonstrate that HFE and Tf

bind to an overlapping site on TfR.48

B. Regulation of TfR Expression

Cellular iron metabolism is self-regulated through iron-dependent changes in the abundance of

TfRs, which control iron uptake, and ferritin, which sequesters within the cell iron. Several studies

have shown that a concentration- and time-dependent decrease of their Tf-binding capacity occur-

red, when the cells grown in the presence of iron salt.49–52 The decrease reflected reduction in


receptor numbers elicited by iron load and was associated with an enhanced intracellular ferritin

content.51,53 Conversely, incubation of the cells with iron chelators caused an increase in number of

receptors, which is dependent on an enhancement of the rate of receptor synthesis, and reduced the

intracellular content of iron, and therefore of ferritin.

The molecular mechanism underlying the regulation of TfR gene expression by iron has been

generally accepted. The regulation is largely posttranscriptional and is mediated by specific mRNA-

protein interactions in the cytoplasm. The 3 0-untranslated region of receptor mRNA contains a

series of five hairpin stem-loop structures required for iron-dependent regulation.54 The stem-loop

structures called iron-responsive elements (IREs) are recognized by trans-acting proteins, known

as iron-regulatory proteins (IRPs),55 that control the rate of mRNA translation or stability.56,57

Two closely related IRPs (IRP-1 and IRP-2) have been identified to date.55,58 Both display IRE-

binding properties under conditions of iron deprivation. IRP-1 has been regarded as a bi-functional

‘‘sensor’’ of iron, switching between RNA binding and enzymatic activities as aconitase depending

on cellular iron status.59–61 In iron-depleted cells, IRP-1 inhibits translation of ferritin by binding

to IREs of ferritin mRNA located in the 5 0-untranslated region.62,63 Binding of IRP-1 to IREs in the3 0-untranslated region of transferrin receptor mRNA stabilized this transcript,56,64,65 thus increase

cellular iron uptake and availability. When iron is high, IRP-1 is enzymatically active and no longer

binds well to the IRE-hairpins, which leads to the degradation of TfR mRNA, thus the inverse effect

ensues.64,65 IRP-2 binds specifically to all known mRNA IREs with an affinity equally as high as

that of IRP-1.58,66 The two proteins are encoded by separate genes. However, IRP-2 is enzymatically

inactive.66 The IRPs both respond to iron, but via different pathway. IRP-1 is post-translationally

converted between active and inactive RNA-binding forms,67 while IRP-2 is induced following iron

starvation through renewed synthesis of stable IRP-2 protein and its inactivation by iron reflects

degradation of IRP-2 by a translation-dependent mechanism.66,68

The signals other than iron levels, such as nitric oxide and oxidative stress, can also regulate

IRPs and modulate cellular iron metabolism.67,69 Nitric oxide and H2O2 produced from oxidative

stress activate IRP-1 by a cycloheximide-insensitive posttranslational mechanism,70 whereas IRP-2

activation by nitric oxide requires de novo protein synthesis.71 The activation of IRP-1 by nitric

oxide closely resembles the pattern of activation observed in iron-deficient cells. The activation

of IRP-1 by H2O2 is different and may involve additional cellular activities probably by accele-

rated cluster removal rather than simply attack on IRP-1. Detailed information can be found in a

review.67

The expression of TfRs is also regulated through the status of cellular proliferation. Generally,

cells undergoing multiplication markedly increase their receptor numbers, while nonreplicating

cells have a stable iron balance. Cell proliferation-associated induction of TfR express could

be mediated by mitogens,72 which modulate various protein kinase activities, through either the

activation of gene transcription or the stabilization of mRNA.73,74 TfRs are also expressed on

several types of nondividing cells such as reticulocytes, trophoblasts, hepatocytes, and tissue

macrophages, suggesting that the relationship between TfR expression and cell activation is not

a generalized phenomenon.75

C. Transferrin Receptor 2 (TfR2)

Recently, a new TfR-like family member, TfR2 has been cloned and sequenced.76 TfR2 shared a

45% identity and 66% similarity in its extracellular domain with TfR. Two transcripts have been

identified. The a-transcript product is a transmembrane protein that is primarily expressed in the

liver of humans and mice,76,77 and the TfR2 b-transcript is the result of alternative splicing.

Its product may be an intracellular protein and distributed widely, but is expressed at low level.76

TfR2 has a similar function to TfR with respect to Tf binding and Tf-mediated iron uptake. Both

230 * LI AND QIAN

Figure 3. Proposed model for binding of serum transferrin to transferrin receptor. The surface of TfR is shown predominantly in

whiteand those incontact with thedocked transferrin inprotease-like, apical, andhelicaldomains colored in red, green, and yellow,

respectively.The N-lobe (N1and N2) of rabbit transferrin are colored in orange and red, the C-lobe (C1and C2) in blue and purple,

respectively.The positionof humanand rabbit N-linked glycosylation sites is indicatedby white andblackasterisks.Themovement

of the N2 and C2 domains upon Fe3þ

release is denoted with white arrows. (Reprinted with permission from Lawrence CM, et al.

Crystal structure of the ectodomainofhumantransferrin receptor. Science1999;286:779^782.� 1999 American Association for the

Advancementof Science.42)

Figure 4. The pathway of cellular uptake of iron from transferrin via transferrin receptor mediated endocytosis. Differic transferrin

is bound by the membrane-bound transferrin receptor and internalized via receptor-mediated endocytosis into endosome.

Uponacidification, iron is releasedand thentransportedby the irontransporter DMT1into the cytoplasm. Apo-transferrinand trans-

ferrin receptor are then recycled back to the cell surface. Iron that enters the cell can be used for metabolic functioning or stored

in ferritin. (Reprinted fromAndrewNC.Disorders of ironmetabolism.N.Engl JMed1999;341:1986 1995 with permission.81)


TfR 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.78 However, the affinity of TfR2 for iron-loaded

Tf is 25-fold lower than that of TfR for Tf.79 The pattern of expression and regulation of TfR2 is

distinct from TfR. TfR2 expression does not show the iron-dependent post-transcriptinal regulation,

and instead may be regulated by the cell cycle or cellular proliferation status.78 The difference

between TfR and TfR2 may partially be attributed to the fact that TfR2 mRNA lacks the iron-

responsive elements.76 TfR2 has been thought to be critical for maintenance of iron homeostasis,

since a homozygous nonsense mutation in TfR2 has been identified as the cause of a form of

hemochromatosis that is not linked to the mutation of HFE.80

4 . C E L L U L A R I R O N U P T A K E V I A T R A N S F E R R I N -R E C E P T O R - M E D I A T E D E N D O C Y T O S I S

A. Iron and Diseases

Iron is essential for almost all organisms, fulfilling a variety of biological functions. Examples

include the transport, storage, and activation of oxygen transport; energy production; cell proli-

feration and a range of catalytic processes. However, iron is potentially toxic if it reacts with oxygen

to generate toxic free radicals that attack cellular membranes, proteins, and DNA. Organisms deal

with this problem by tightly regulating the concentration of iron in their internal fluids. In humans

and other mammals, no effective excretory pathway exists. Instead, the cells lining the gut and the

enterocytes control iron absorption and its export from the enterocytes to the blood.81 The normal

human adult has 35–45 mg of iron/kg of body weight and more than two thirds is incorporated into

hemoglobin in developing erythroid precursors and mature red cells.81 Dietary iron absorption is

normally tightly linked with body utilization through the sensing of body iron statues in the

proximal small intestinal.82,83

Defects in iron absorption and utilization lead to iron deficiency, overload disorders, and certain

neurodegenerative disorders.81,84 More than half a billion people worldwide suffer adverse effects

as the result of iron deficiency (iron-deficiency anemia and anemia of chronic inflammation).81

Hereditary hemochromatosis (HH) is a common disorder of iron overload,85–87 which is the most

common genetic disorder, affecting between one in 200 and one in 400 Caucasian individuals.88

The disease is characterized by inappropriate control of intestinal iron absorption, resulting in

excessive accumulation of iron in organs such as the liver, heart, and pancreas, eventually leading

to multi-organ dysfunction.89 Certain neurodegenerative diseases including Hallerorden-Spatze

syndrome (HSS), Parkinson’s Disease (PD), Alzheimer’s Disease (AD), and Huntington’s Disease

(HD) have been shown to be associated with elevated levels of iron and oxidative stress in

the brain.90–93 Although the role of brain iron imbalance in the development of neurodegenerative

diseases is so far unknown, brain iron malregulation appears to be an initial cause of neuronal death.

It has been speculated that disruption in the expression of brain iron transport proteins is probably

one of the important causes.94,95

B. Transferrin-Receptor-Mediated Iron Uptake

Most, if not all, mammalian cells are capable of taking up iron by receptor-mediated endocytosis

of diferric transferrin bound to the transferrin receptor. Our knowledge concerning cellular iron

transport has been markedly advanced by the recent discoveries of several genes such as HFE,96

associated with hereditary hemochromatosis, and divalent metal transporter (DMT1/Nramp2),

a membrane iron transporter.97,98 Those proteins are likely to play an important role in the trans-

ferrin cycle. It is generally accepted that the process of transferrin endocytosis can be disting-

232 * LI AND QIAN

uished into six steps, i.e., binding, internalization (endocytosis), acidification, dissociation, and

reduction, translocation, and cytosolic transfer of iron into intracellular compounds such as ferritin

or heme.10,11,35,99

Cellular uptake of iron is initiated by the binding of diferric-transferrin to specific TfRs on

the outer face of the plasma membrane. The binding is apparently a simple chemical event not

dependent on metabolic energy.100 It has been thought previously that each TfRs binds to two

molecules of Fe2-Tf with a high affinity at physiological pH (7.4). However, recent studies have

implied that the interaction of Fe2-Tf with TfRs is modulated by HFE,48,101–105 the protein mutated

in hereditary hemochromatosis. It has been demonstrated that HFE and Fe2-Tf can bind simul-

taneously to TfR to form a ternary complex consisting of one Fe2-Tf and one HFE bound to a TfR

homodimer,48 and that HFE inhibits the TfR- Fe2-Tf interaction by binding at or near the transferrin-

binding site on TfR.48,101,103 Furthermore, HFE binds TfR tightly at the pH of the cell surface,

but not at pH 6, suggesting that HFE can be dissociated from TfR in acidified endosomes.106,107

Indeed, HFE association with TfR has been shown to negatively regulate Tf-mediated iron uptake

in transfected cells.103,104

After endocytosis via clathrin-coated pits, which eventually bud from the plasma membrane

as membrane-bound vesicles or endosomes, the Tf�TfR�HFE or Tf2�TfR complexes are routed into

the endosomal compartment (Fig. 4). Upon maturation and loss of the clathrin coat, the endosome

becomes competent to pump protons in a process energized by adenosine triphosphate (ATP)

and endosomal lumen is rapidly acidified to a pH of ca. 5.5.108,109 Iron is released from Tf.

The mechanism through which iron releases is still not clear, although the acidification has been

though to be essential for the efficient release of iron. A study showed that TfR may facilitate the

release of iron from differic transferrin at a low pH,110 presumably that transferrin receptor

also changes conformation at low pH and the motion of apical domain or other domains of TfR can

force one or both lobes of transferrin into an open conformation, thus facilitating release of iron.42

The free Fe(III) released to endosomes is reduced to Fe2þ on the cis-side of the endosomal

membrane probably mediated by oxidoreductase.111 How iron crosses the endosomal membrane to

enter the cytosol remained unknown until the discovery of DMT1,97,98 which has been shown to be

an apical transmembrane iron transporter that actively transports reduced dietary iron into intestinal

enterocytes. DMT1 has also been demonstrated to be essential for the transport of iron out of

the transferrin cycle endosome, i.e., from the endosomal membrane to the cytosol.112,113 DMT1 is a

new proton-couple metal-ion transport protein with putative 12 transmembrane domains, among

which the transmembrane domain 4 has been implicated in its function and mutation of G185R

in the transmembrane domain 4 disrupts its function.114 However, nothing is known about the

mechanism through which iron is transported and further studies are urgently needed. Once in

the cytosol, iron is utilized as a cofactor for aconitase, the cytochromes, RNA reductase, and heme,

or it is stored as ferritin. After release of iron into the endosome, the resultant apo-Tf�TfR complex

is recruited through exocytic vesicles back to the cell surface and apo-Tf is released at extracel-

lular fluid due to its low affinity at pH 7.4, thereby completing an elegant and efficient cycle.

The behavior of iron in the brain is a topic that is presently causing excitement. This is largely

due to the finding of the existence of abnormally high levels of iron and oxidative stress in neuro-

degenerative disorders.91,115,116 As in other tissues outside of the brain, Tf/TfR-mediated endo-

cytosis is probably the main mechanism of iron uptake by most brain cells and Tf-bound iron is the

major iron transport form in the brain since brain neuronal cells, oligodendrocytes and brain

capillary endothelium have the ability to express TfR.117,118 However, TfR expression is likely not

the only factor determining iron uptake by brain cells, nor is Tf the only transporter of iron in

the brain. Recently, with the discovery of many proteins that relate to brain iron regulation,

the mechanisms of iron metabolism and homeostasis in the brain are thought to be, at least in part,

different from or more complicated than those in tissues and cells outside of the brain.94,95 Apart

from Tf/TFR, other proteins such as lactoferrin receptor, melanotransferrin, ceruloplasmin, and


divalent metal transporter (DMT1) are likely to play a role in physiological iron transport in the

brain. Disrupted expression of any of these proteins may be connected with excessive accumulation

of brain iron in neurodegenerative diseases.94,95

5 . T R A N S F E R R I N A S A M E T A L L O D R U G C A R R I E R

A. Therapeutic Metal Ions: Bi, Ru, and Ti

Bismuth has been used in medicine for more than two centuries. Various bismuth complexes

have been used to treat a wide range of diseases such as syphilis, hypertension, infections, skin

conditions, and gastrointestinal disorders.119 Currently, three bismuth compounds have been the

most commonly used worldwide—bismuth subsalicylate (BSS, Pepto-Bismol1; the Procter &

Gamble Company, Cincinnati, Ohio) for the prevention and treatment of diarrhea, and dyspepsia;

colloidal bismuth subcitrate (CBS, De-Nol1; Gist Brocades, Delft, The Netherlands) for the

treatment of peptic ulcers and ranitidine bismuth citrate (GlaxoWellcome, Tritec1 and Pylorid1),

which combines the antisecretory action of ranitidine with the mucosal protectant and the bacte-

ricidal properties of bismuth.120 The chemical properties and structures of bismuth containing drugs

have been intensively reviewed.121–124

Despite the widespread use of bismuth compounds in medicine, its mechanism of action,

transportation and toxicity (especially encephalopathy) are still poorly understood. A gel filtration

study of human blood after incubation with bismuth subgallate showed an association of bismuth

with high molecular mass ligands,125 but it is not clear which protein could possibly be the target.

Albumin, the most abundant plasma protein, was previously speculated to be a potential target.126

However, a recent study of competitive binding of transferrin and albumin in aqueous solution and

in blood plasma showed that bismuth binds preferentially to transferrin instead of albumin, and

binding induced conformational changes, e.g., from the lobe-open to the lobe-closed form which is

crucial for transferrin receptor recognition.127 Therefore transferrin may act as a carrier to deliver

bismuth into the cells. Several studies have shown that bismuth binds strongly to transferrin and

lactoferrin in the specific iron binding sites with affinities similar as iron.128–131 Bi-transferrin and

Bi-lactoferrin can be recognized by BeWo placental cancer cells and IEC-6 rat intestinal cells,

respectively and both interfere with iron transportation.132,131 This provides an evidence that

bismuth is likely transported via a similar mechanism as iron, i.e., transferrin receptor mediated

endocytosis. This transportation mechanism may also have implications for bismuth antimicrobial

action. It has been found that the resistance to the inhibitory action of bismuth among Gram-

negative bacteria is inversely related to iron concentration and strongly dependent on the iron

transport mechanism.133 Presumably this is because bismuth blocks the pathway of iron transport

into the bacteria and cuts iron supply required by the bacteria for its growth. Another pathway

may also exist for bismuth transportation such as via thiol-containing ligands since bismuth has

been strongly associated with tripeptide glutathione and the cysteine-rich protein metallothionein.

This has led to an expectation that glutathione and metallothionein may play a role in the transport

and delivery of bismuth in cells and biofluids.134,135 However, further studies are needed to establish

this.

Ruthenium complexes with various ligands such as amine and dimethylsulfoxide exhibit high

anticancer activity in vivo and are potential anticancer agents. They are often active against

metastases but not against the primary tumors.136,137 It appears that Ru3þ is transported in the blood

by transferrin and albumin. Ru3þ complexes were reported bound to both albumin and transferrin

with 80% portion binding to albumin and the remainder to the latter.136,138,139 It required 5 mol

equiv. Ru3þ complex for saturation of albumin, whereas only 2 mol equiv. for saturation of

transferrin. The binding site for albumin was proposed at the surface histidine.138 The X-ray crystal

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structure of human lactferrin-Ru3þ showed that Ru3þ coordinated directly to the imidazole

nitrogen of His253, one of the iron ligands in the iron binding site in the N-lobe, with displacement

of a chloride ligand, while the heterocyclic ligands remain coordinated within the protein.140

Injection of Ru3þ -TF resulted in high tumor uptake of the metal,141–143 which suggests that

transferrin uptake appears to be the more important mode of transport of Ru3þ anticancer com-

plexes to the tumor since elevated level of TfRs were found in many solid tumors than in normal

cells. Transferrin-mediated uptake may lower ruthenium toxicity by preventing it from other

binding or uptake until it has been delivered to the cells. Therefore, Ru-Tf complexes may provide a

new family of less toxic and more effective antitumor agents. Indeed, the transferrin-bound complex

exhibits a significantly higher antitumor activity against human colon cancer cells than the albumin-

bound complex or the Ru3þ complex itself.136,144

Ti4þ 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.145–147 Currently, there

are two titanium complexes, titanocene dichloride and budotitane, now in clinical trials.145,148

Titanocene dichloride is active against a diverse range of human carcinomas, including gastro-

intestinal and breast carcinomas, but not against head and neck cancers. It also exhibits pronounced

antiviral, antiinflammatory and insecticidal activities.149 Apart from the potential therapeutic use of

titanium complexes, an enormous amount of titanium is present in a variety of biomaterials and in

many foods as whitening pigment. Therefore it is likely that titanium enters into the living systems.

However, the pathway through which titanium enters into the cells is still poorly understood.

Formation of titanocene dichloride-DNA complexes has been previously implicated in the

mechanism of antitumor properties of the drug since titanocene dichloride inhibits DNA synthesis

and titanium accumulates in nucleic acid-rich regions of tumor cells after in vivo or in vitro

administration.149,150 However, this has been challenged by a recent study which shows that Ti4þ

does not bind strongly to DNA bases at physiological pH but forms strong complexes with

nucleotides only at low pH values (below 5).151 Thus, a carrier is required to deliver titanium

complexes to tumor cells and to prevent hydrolysis of Ti4þ complexes at neutral pH.146,152

Recently, it has been reported that Ti4þ binds strongly to transferrin in the specific iron binding

sites. Binding induces structural changes in a similar manner as iron, and titanium can be released

at acidic pH values in the presence of citrate or ATP.132,153,154 In addition, Ti2-Tf can block both

membrane binding and cellular uptake of Fe2-Tf into BeWo placental cancer cells. Therefore, it is

likely that transferrin mediates the uptake of titanium from the anticancer drugs into tumor cells and

titanium is then released subsequently due to acidic microenvironment in tumors than in normal

tissue,155 and targets DNA.

B. Diagnostic Radioisotopes: Ga and In

Gallium compounds have been used extensively both in the diagnosis and the treatment of human

cancers.156 67Ga, a low energy gamma emitting radionuclide, is one of the most useful tumor

diagnostic agents available. 68Ga is of growing interest because it is suitable for three dimensional

imaging by position emission topography. Gallium nitrate has been used clinically to treat hyper-

calcemia of malignancy and bone diseases such as bone metastases.157

It has been shown previously that Ga3þ binds to transferrin in the specific Fe3þ binding sites

with a similar affinity, attributed to the similarity between these two metal ions.158 In vivo studies

using 67Ga find that all gallium in blood is present in plasma (with traces in leukocytes) and is tightly

bound to transferrin.159 It is generally accepted that gallium is transported mainly via transferrin

receptor mediated mechanism. Transferrin can enhance 67Ga uptake into EMT-6 tumor cells.160

Gallium-Tf can also be taken up by human leukemic HL60 cells and blocks the activity of the iron-

dependent enzyme ribonucleotide reductase.161–164 Ga3þ is expected to concentrate in tissues

having a high concentration of TfR, LfR or ferritin, such as proliferating tissue including most


tumors, milk, tears, and areas of inflammation. Indeed, good correlations were found between TfR

expression and 67Ga uptake in malignant tissue.161,165,166 The high concentration of gallium in

tumor is the basis for the widespread use of 67Ga imaging as a diagnostic technique for many

malignancies. Gallium can also enter tumor and other cells by a Tf-independent mechanism, which

is probably also used by iron.161,165 This becomes apparent when Tf is in short of supply or saturated

with iron or other metal ions.

Like Ga3þ , the In3þ has also been investigated intensively because of the widespread interest

in its use in radiopharmaceuticals. Two g-emitting isotopes, 111In (t1/2¼ 2.8 days) and 113In (t1/2¼1.7 h) are of interest. 111In containing compounds may be employed in a combination chemo-

therapy/radiotherapy approach to treat neoplasms, or as a tumor-localized source of irradiation and

as a radiolabel for the determination of tumor cell viability.

In3þ , which more closely resembles Fe3þ than Ga3þ , binds to transferrin strongly but slowly

compared with Ga3þ .168 When indium is injected either as an acidic solution or as a weak chelate

such as citrate, more than 95% binds to macromolecular ligands, which appear to be trans-

ferrin,169,170 although a recent study showed that albumin may also be responsible for the binding

and transport of indium in serum.171 When a 111In compound is administrated in a strongly

complexed form, there will be no binding to transferrin. The transferrin receptor mediated uptake

of indium is less effective in comparison with iron. The binding affinities of In2-Tf and Fe2-Tf to

the transferrin receptors on reticulocytes are very similar. However, unlike iron, transferrin-bound

indium remains bound to the cell membrane, there is minimal transfer into the cell or incorporation

into heme.172 Similarly, it has been shown that In2-Tf can bind to the transferrin receptors at the

placenta, but there is no actual transport of indium across the membrane.173 The reason appears to be

unknown. Despite many problems associated with receptor mediated uptake of indium, it was found

that indium still tends to localize in tissues with large numbers of transferrin receptors.174 Much

needs to be learned about the role which transferrin plays in the delivery of indium into the cells.

6 . T R A N S F E R R I N C O N J U G A T E S I N D R U G A N D G E N E D E L I V E R Y

Targeted drug delivery has gained recognition in modern therapeutic and attempts are being made

to explore the potentials and possibility of cell biology related bioevents in the development of

specific, programmed, and target-oriented systems. Among those, receptor-mediated cellular events

have received major attention in the past few years. A number of reviews in this field are

available.3,5,6,175 Transferrin, either in the form of drug conjugates, hybrid systems with marco-

molecules or as liposome-coated systems, has been used as a carrier or targeted ligand to delivery

anticancer drugs, drug containing liposomes, proteins, and genes to primarily proliferating cancer

cells that overexpress transferrin receptors.

A. Transferrin Conjugates in Drug Delivery

Cardiotoxicity and development of resistance towards cytotoxic drugs like doxorubicin

(Adriamycin1) constitutes a major problem in cancer chemotherapy. Various approaches have

been devised to circumvent these limitations, amongst which is the attachment of cytotoxic drugs

to suitable carrier proteins, such as transferrin, that accumulate in tumor tissue. Doxorubicin

conjugated with transferrin through glutaraldehyde crosslinking technique was demonstrated to be

selectively cytotoxic towards a variety of cultured cell lines, for example, leukemic cells, the murine

L929 cell, a human bladder transitional cell carcinoma cell RT-4 and a human breast cancer cells

MCF-7.176–180 A preliminary clinical study showed the therapeutic usefulness of this conjugate in

the treatment of certain leukemias.181 Moreover, the doxorubicin-transferrin conjugate was shown

to exert a cytotoxic effect in partially (KB-8-5) or in highly multidrug-resistant (KB-C1 and KB-V1)

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cells and the conjugate exhibited a lower IC50 concentration than doxorubicin in all KB cell lines

examined.182 Similarly, the conjugation of doxorubicin to gallium transferrin can overcome mul-

tidrug resistance in breast cancer cells (MCF-7) and the conjugate accumulates in the cytoplasma

and nucleus of both the multidrug resistance and parental MCF-7 cells.176 The doxorubicin-Tf

conjugate exerts its cytotoxic effects probably through a transmembrane mechanism,176,183,184

different from doxorubicin, which enters the cell cytoplasm and enhances the synthesis of

P-glycoprotein, a protein function as a pump and is capable of removing doxorubicin from the

cytoplasma.185 Chlorambucil (leukeran), another anticancer drug used clinically against chronic

lymphatic leukemia, lymphomas and advanced ovarian and breast carcinomas, is limited by its toxic

side effects. The conjugation of chlorambucil with transferrin through an acetaldehyde carboxylic

hydrazone bond exhibited IC50 values approximately 3–18-fold lower than those of chlorambucil

in the MCF7 mammary carcinoma and MOLT4 leukemia cell line. And preliminary toxicity studies

in mice showed that this conjugate can be administrated at higher doses compared with unbound

chlorambucil.186 Transferrin- mitomycin C (MMC), the chemotherapeutic DNA crosslinking

agent, has been demonstrated to be a useful hybrid as a receptor-mediated targeting system.187–189

The Tf-MMC conjugate bound and was internalized into the human hepatoma cell line HepG2 cell,

normal cultures rat hepatocyte, human leukemia cell line HL60 cells and Sarcoma 180 cells.

The proliferation of the HepG2 and HL60 cells was inhibited by Tf-MMC in vitro.187,188 The

transferrin-mediated endocytosis was also used to attempt to import bioactive marcromolecules

(e.g., anti-tetanus fragments) into cells using an acid-labile transferrin conjugate,190 to delivery

insulin in cultured human enterocyte-like Caco-2 cells and in streptozotocin induced diabetic rats

using insulin-Tf conjuagate linked by a disulfide bond.191 Recently, a novel approach for the

biological delivery of therapeutic peptide has been achieved by incorporating the sequence of the

peptide into the structure of a natural transport protein, such as transferrin.192 The mutant proteins

retained native transferrin function and the inserted peptide sequence was surface exposed and could

be cleaved easily. This novel approach is potentially useful for developing therapeutic agents for a

broad spectrum of diseases.

Liposomes, consisting of one or more concentric phospholipid bilayers, have shown promise

in the introduction of chemotheraputic agents with reduced toxicity, extended longevity and poten-

tial for cell-specific targeting. A liposomal carrier system, which was produced by using small

unilamellar liposomes made of pure phospholipids chemically cross-linked to human transferrin,

was reported to interact specifically with leukaemia HL60 cells and the conjugate was subsequently

internalized by active receptor-mediated endocytosis.193 Transferrin-coulped liposome, in which

transferrin was coupled to the distal ends of liposome polyethylene glycol, was shown to target

specifically to C6 glioma in vitro. Doxorubicin encapsulated within transferrin-coupled liposomes

could enhance the uptake of free doxorubicin via the receptor-mediated mechanism.194 A study

of the uptake of liposome and the antiproliferative effect of liposome-entraped alpha-interferon

(alpha-IFN) against murine bladder tumor cell MBT2 showed that when liposome conjugated with

transferrin-polylysine (TFPL), cell uptake of TFPL-liposome was markedly enhanced in a dose-

dependent manner. There was also a strong correlation between antiproliferative activity and uptake

of liposome by the tumor cells, indicating that TFPL-liposome promotes intracellular delivery of

alpha-IFN and enhances the effect of alpha-IFN against MBT2 cell growth.195 Tf-pendant type

immunoliposome (TF-PEG-ILP) was shown to have a higher uptake to K562 cells in vitro compared

with non-targeted-liposomes. And the initial binding localized on the cell surface at 4�C and

internalization by endocytosis was confirmed upon raising the temperature to 37�C.2 The TF-PEG-ILP, examined in the B16 melanoma-bearing mice, exhibited prolonged circulation time, low liver

uptake and concomitantly high accumulation into the tumor tissue and longer residence.2

Liposomes conjugated with anti-transferrin receptor have also been used for specific drug delivery.

A liposome-immoblized Anti-Tac (a monoclonal antibody against the IL-2 receptor) and Anti-TfR

(a monoclonal antibody against transferrin receptor) was compared for specific binding,


internalization and intracellular drug delivery to adult T-cell leukemia.196 It was found that there

was a better growth inhibition profile of Anti-TfR-coupled liposome over Anti-Tac-coupled lipo-

somes bearing methothrexate-g-aspartate, a liposome-dependent cytotoxic drug.196

B. Transferrin Conjugates in Gene Delivery

The specific delivery of therapeutic genes to defined target cell populations is a major goal of

gene therapeutic strategies. Viral vectors generally facilitate highly efficient transfer and expres-

sion of foreign genes, but attempts to modify their target cell specificity have proven difficult.197

Viral vectors can be immunogenic, cytopathic or recombinogenic; for example, adenoviral vectors

can induce host immune response, thus rendering their repeated applications.198 Non-viral vectors

including molecular conjugates and cationic liposomes are being exploited as promising alter-

natives. However, gene delivery employing these non-viral vectors suffers from low transfection

efficiency and much effort has been made towards improving the transfection efficiency.

Molecular conjugates is a synthetic gene delivery vector composed of nucleic acids condensed

with polycations (such as polylysine, polyethyleneimine) that can be cross-linked to a ligand for cell

targeting. After binding to the cell surface, conjugates are internalized, and a small fraction of them

escape from the endocytic network and translocate to the nucleus, where genes within the DNA of

the conjugates are expressed. Transferrin has been used as a general targeting molecule to direct

DNA to rapidly dividing cells. Transferrin-polylysine and transferrin-protamine conjugates have

been shown to be efficient carriers for the introduction of genes into many cells such as human

leukemic cells K-562 and hematopietic cells.199–202 Such a delivery system was also shown to be

efficient for the selective delivery of oncogene-targeted antisense oligodeoxynucleotides. It was

shown that exposure of HL-60 cells to the myb antisense/transferrin-polylysine complex resulted in

rapid and profound inhibition of proliferation and loss of cell viability much more pronounced

than that occurring in cells exposed to free myb antisense oligodeoxynucleotides.203 Similarly,

transferrin-polyethylenimine (PEI) conjugates have also been demonstrated as vectors to trans-

fer therapeutic DNA into cells.204–206 Systemic application of transferrin-PEI-DNA into A/J mice

bearing subcutaneously growing Neuro2a tumors via the tail vein resulted in preferential luciferase

reporter gene expression in distant tumors.204 However, one limit to successful receptor-mediated

gene delivery is the exit of endocytosed material from the endosome. Different strategies have been

developed to ensure the release of DNA from internal vesicles. Addition of chloroquine during

transfection, preventing acidificatin of endosomal and lysosmal compartment, is one measure to

ensure better survival and transfer of DNA into the nuclear compartment.201,202 Coupling of

adenovirus to transferrin-polylysine/DNA complexes is another method to enhance receptor-

mediated gene delivery.207–209 Therefore, the development of specific mechanism to effect release

from the endosome in combination with gene transfer by the receptor-mediated endocytosis path-

way will increase the utility of this delivery system by allowing high levels of gene expression in

target cells.

Cationic liposomes complexed with DNA have been used extensively as non-viral vectors for

the intracellular delivery of reporter or therapeutic genes in culture and in vivo.210 It is believed that

the majority of DNA complexed to cationic liposomes is taken up through endocytosis, followed by

its release from an early endosomal compartment. However, poor transfection efficiency is the major

drawback of these vectors. In addition, application of cationic lipid-DNA complexes (lipoplexes)

in vivo is also limited by the inhibition of serum. Association of transferrin with cationic liposome-

DNA complexes, in particular the negatively charged ternary complexes, significantly overcame the

inhibitory effect of serum and enhanced the transfection efficiency in many cell lines including

HeLa, K562 cells and lung carcinoma cells Calu3, H292 cells.211–214 This strategy was also effec-

tive in enhancing transfection in epithelial and lymphoid cell lines, as well as human marcophages,

especially with the use of optimized lipid/DNA (þ/�) charge ratios.215 Similarly, using the

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transferrin-liposome system, p53 gene has been successfully transfected into a head and neck

squamous carcinoma JSQ-3, and the introduced p53 was able to sensitize the transfected JSQ-3 cells

to ionizing radiation, which may provide a more effective treatment for head and neck cancer.216

C. Transferrin Receptors in Drug and Gene Delivery to the Brain

The delivery of non-lipophilic compounds to the brain is severely limited by the tightly apposed

capillary endothelia cells that form the blood-brain barrier (BBB). However, brain capillary

endothelia cells do possess specific receptor-mediated transport mechanisms that potentially can be

exploited as a means to delivery therapeutic molecules to the brain. The antibodies that bind to the

transferrin receptor have been shown to selectively target BBB endothelium due to the high levels

of transferrin receptor expressed by these cells.217,218 Therefore, these antibodies are potential

carriers for the delivery of therapeutic agents to the central nerves systems (CNS). Amongst these

antibodies, the OX26 monoclonal antibody against the rat transferrin receptor is the most widely

used antibodies in the delivery therapeutic agents to the brain. It has been reported recently

that immunoliposomes (antibody-directed liposomes), when conjugated with the OX26 monoclo-

nal antibody against the rat transferrin receptor, showed potentials for brain drug and gene

delivery.219,220 Small molecule drugs or an exogenous plasmid DNA has been incorporated into

the interior of neutral liposomes, which are pegylated with PEG of 2,000 Da molecular mass.

A thiolated antibody, the OX26 murine mAb to the rat transferrin receptor was coupled to the

terminal end of PEG 2000. Successful delivery of small molecule drugs, such as the antineoplastic

agent daunomycin, to the rat brain has been achieved.219 Similarly, widespread gene expression

in brain after noninvasive i.v. administration of a 6–7-kb expression plasmid, encoding either

b-galactosidase or luciferase, has been achieved by this method.220 By designing conjugates

between OX26 and therapeutic agents, such as low molecular drugs (methotrexate), neuropeptides

(vasoactive intestinal peptide), polyamide nucleic acids, proteins (never growth factor NGF),

which can be transcytosed via the transferrin receptor to the brain side of the blood-brain barrier,

the effective concentration of drugs delivered to the brain has been markedly increased compared

to the intravenous administration of the drugs alone.221–226

In addition to the chemical conjugation, attempts have also been made to couple the thera-

peutics to the OX26 antibody using the avidin/biotin systems to promote coupling of biotin and

biotinylated drugs to brain transport vectors.227–229 A novel Ab-avidin fusion protein (Ab gene-

tically fused to avidin) was constructed to deliver biotinylated compounds across the blood-brain

barrier, and the fusion protein exhibited superior [3H]biotin uptake into brain parenchyma in

comparison with the chemical conjugate OX26-SA (Ab chemically conjugated to streptividin).228

Furthermore, brain uptake of the HIV antisense drug increased dramatically when it was bound to

the fusion protein.228 Similarly, a single chain Fv antibody (of OX26)-streptavidin fusion protein

could facilitate the attachment of biotinylated drugs to the antibody vector.229 Therefore, this kind of

fusion protein is potentially important for effective delivery of biotinylated compounds across the

blood–brain barrier for diagnosis or therapy of a broad range of central nervous system disorders.

7 . C O N C L U S I O N S A N D P E R S P E C T I V E S

Transferrin/transferrin receptor mediated endocytosis is a major pathway for entry of iron into

mammalian cells. Intensive studies have been directed to understand this process at the molecular

level for the past few decades. Particularly the identification of HFE as a hereditary hemo-

chromatosis and DMT1 as iron transporter represents a major breakthrough and provides an insight

into the mechanism of iron absorption, transport and the cellular regulation of iron metabolism.

The HFE binds to transferrin receptors and negatively modulates the receptor’s activity. It is also


clear now that DMT1 is responsible for transporting of Fe2þ from the endosomal membrane into

the cytoplasm. However, much needs to be learned before the details of this process are fully

understood. Nothing is known about how DMT1 transports Fe2þ and why mutation of G185R in the

transmembrane domain 4 impairs its function. It should also be interesting to study the newly

discovered transferrin receptor, TfR2, and its function, the regulation of its expression, structure and

its interaction with transferrin.

Transferrin mediation provides a specific pathway for the delivery a variety of therapeutic

and diagnostic metallodrugs. It is known that Ga3þ and Ru3þ complexes can be delivered into

cells by transferrin. Ti4þ and Bi3þ are also bound transferrin in the specific binding sites and their

transferrin complexes can be recognized in cultured cells. However, there is a lack of clinical

support and more work is needed to clarify this. It is also not clear whether transferrin is able to

delivery In3þ to the cells. Moreover, there is much potential to be exploited for the other members

of the transferrin family as drug mediators. The mediation processes by these proteins such as

lactoferrin and melanotransferrin are currently poorly understood. More detailed understanding

the metal mediations would enable us to design new metal-based drugs and therapeutic agents and

also lead to a better and safer use of the drugs.

Transferrin/transferrin receptor mediated cellular events have also been exploited as carrier

systems to delivery therapeutic drugs and genes into malignant cells that overexpress transferrin

receptor. By designing transferrin conjugates with anticancer drugs, proteins and DNA, which

condensed either by polycations (e.g., polylysine) or carried by cationic liposomes, achievements

have been made to deliver these therapeutic agents into target cells or tissues. Moreover, anti-

transferrin receptor-drug conjugates could deliver drugs and genes across the blood–brain barrier

for treatment of broad spectrum of central nerves system diseases. In gene therapy, exogenous DNA

that has been coupled to transferrin can be targeted to proliferating and hemopoietic cells and

internalized via endocytic pathways for efficient delivery and expression of foreign genes in the

desired cell nucleus. However, its clinical application is restricted by a number of factors,

particularly the low targeting and transfection efficiency. Much needs to be investigated before this

system can be successfully used clinically. These may include chemically modifying the system,

such as optimizing parameters affecting surface binding and associations and developing a specific

mechanism to effect release therapeutic genes from the endosome into the cytosol. More intere-

stingly, an approach using protein engineering to incorporate the sequence of therapeutic proteins

or peptides into the structure of natural transport protein such as transferrin, would offer potentials

for developing new therapeutic agents in the future.

A C K N O W L E D G M E N T S

The studies in this laboratory were supported by The Hong Kong Research Grants Council (A/C:

BQ-445), The Hong Kong Polytechnic University ITS Grants (A/C: G.12.xx.93A2) and the Post-

doctoral Fellowship Scheme (A/C: G-YW47). We are grateful to Dr. Zuccola HJ of Harvard

University for supplying X-ray coordonates.

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Hongyan Li was born in north China, received her B.Sc. degree in 1988 and M.Sc. degree in 1991 from the

Chinese Academy of Sciences, and from 1991 to 1994 was a research assistant at the Nanjing Institute of Soil

Science of the Chinese Academy of Sciences, PRC. After two years as a research assistant with Prof. Peter

Sadler at Birkbeck College, University of London, she began her Ph.D. studies on inorganic biochemistry under

the direction of Prof. Peter Sadler at the University of Edinburgh, UK, and obtained her Ph.D. degree in 2000.

Currently she is working as a Post-doctor fellow in the laboratory of Dr. Zhong Ming Qian, Department of

Applied Biology and Chemical Technology. The Hong Kong Polytechnic University.

Zhong Ming Qian was born in Suzhou, PRC, and received his M.D. degree in Pathophysiology at Beijing

University Medical College (Beijing Medical University) of PRC in 1981 and Ph.D. degree in Physiology at the

University of Western Australia in 1993. His doctorate research on the mechanisms of iron uptake by

mammalian cells was under the direction of Prof. Evan Morgan. He took a position as a lecturer in the

Department of Applied Biology and Chemical Technology at the Hong Kong Polytechnic University in

November 1993, and is now an Associate Professor at the same University. Currently he is also Associate

Director of the University’s newly established Shenzhen Institute of Natural and TCM-based Products. His

current research interests are in the areas of brain iron metabolism, metal transport proteins, and the role of

brain iron in neurodegeneration.

250 * LI AND QIAN

Documents

Endogenous carriers and ligands in non-immunogenic site-specific drug delivery