Recent Developments in Drug Delivery to the Nervous System
Dusica Maysinger, Radoslav Savic, Joseph Tarn, Christine Alien, and Adi Eisenberg
McGill University, Montreal, Quebec, Canada
NEUROACTIVE AGENTS AND THEIR
DELIVERY TO THE CNS AND PNS
In the past decade, the contribution of the material sciences to
drug delivery to the brain was realized mainly through the
use of nonbiodegradable cylinders for intracerebral implantation of genetically engineered cells, or through the
use of polymeric matrices that contained drugs. More recently, further progress has been made in the arena of prodrugs or conjugates that can exploit existing transport systems. An understanding of the basic mechanisms of the
blood-brain barrier (BBB) transport biology provides a
broad platform for current and future nervous system drug
targeting strategies. In general, current approaches are either
invasive (e.g., neurosurgical), pharmacological (e.g., by
applying lipid carriers, liposomes, or different kinds of
nanoparticles), or physiological (e.g., by taking advantage of
normal endogenous pathways of carrier-mediated transport or
receptor-mediated transport). Lipid-soluble molecules that
have molecular mass under 500 daltons access the brain via
lipid-mediated transport, but hydrophilic molecules such as
peptides are mainly transported via receptor-mediated
endocytosis. The main concepts of and underlying
strategies for the administration of clinically relevant
growth factors to the PNS, (1), and to overcome the BBB
in the CNS, are summarized in several reviews (2-6).
A. Problems with Hydrophilic Agents
Although the surface area of the BBB in the human brain is
large [approximately 20 m2 (7)], small hydrophilic molecules
cannot access the brain in pharmacologically adequate
amounts when administered systemically or orally. This
applies also to small peptidomimetic agents such as nerve
growth factor (NGF)-mimetics (8) or neurotensine
mimetics (9); hence effective delivery of these agents will
require a drug delivery and targeting vehicle, or they
should be conjugated to a BBB-targeting system. Development of novel drug delivery strategies requires adequate
biological models to test their suitability. In vitro models
include (i) primary cultures, (ii) immortalized neuronal,
glial, and cerebromicrovascular endothelial cultures, (iii)
hippocampal immortalized neuronal cultures (10,11), (iv)
human cerebromicrovascular endothelial cell lines as a
model of the BBB (12), and (v) more complex cocultures
of neuronal and glial cells (13). In addition to these in vitro
models, a number of in vivo model systems have been employed for testing neuroactive agents and their delivery
systems. Rodent models, although indispensable and most
commonly used to investigate neurological diseases, have
limitations: (i) In general, they show some, but rarely all, of
the pathological features of human neurological diseases;
(ii) the time course of the progression of the disease is
limited due to the difference in life span between two
species; and (iii) tests for verbal communication skills cannot
be applied. A number of neurological disorders are associated with either a lack of neuroactive peptides (e.g.,
growth factors, neurotrophins) or malfunctioning of their
receptors (defective binding between receptor and ligand,
impaired internalization and transport of the receptor-ligand complex, or impaired signaling pathways downstream from the receptor site) (14-20). For example, abnormal growth factor levels in the CNS and/or PNS have
been associated with Alzheimer's disease, Parkinson's disease, Huntington's disease, and diabetic neuropathy (2124). Results from preclinical studies employing both in
vitro and in vivo models discussed above suggest that individual growth factors (as representatives of hydrophilic
molecules) can indeed correct, prevent, or delay some of
the pathological features characteristic of diabetic neuropathy,
Alzheimer's, Parkinson's, and Huntington's diseases.
However, due to the complexities involved in these pathologies, a simple replacement therapy employing drug delivery
systems containing individual hydrophilic neurotherapeutics will most likely be used in conjunction with gene
therapy and/or stem-cell therapies.
B. Problems with Lipophilic Agents
In general, lipophilic agents have little difficulty in penetrating cell membranes, including those of the BBB. The
more lipophilic a drug is, the more readily it will cross the
BBB and reach the brain. Thus the main problem with
these agents lies not in their permeability but rather with
aspects of (i) specificity and selectivity of action in the
brain, (ii) neurotoxicity, and (iii) poor solubility and unfavorable pharmacokinetic properties. Some of these problems can be solved, at least partially, by incorporating the
drug into a carrier polymer so that the release is slower
and the toxicity is reduced. An attempt to increase the specificity and selectivity of neuroactive lipophilic drugs has
been made by conjugation of the drug either with a specific
ligand or with an antibody toward a protein specifically
expressed at the cell surface (3). More recently, a class of
lipophilic compounds, neurosteroids, i.e., steroids known to
be particularly effective in the nervous system, were
found to influence the brain's functions significantly,
memory in particular. These agents do not have a problem in
crossing the BBB or in specifically binding to their receptors. Studies by Toran-Allerand and colleagues showed
that estrogen receptors are localized in central cholinergic
neurons, and that signaling pathways activated by growth
factors can be also activated by estrogens (25,26).
Neurosteroids have been tested in several models (27), and
numerous studies are currently underway to provide a
proof of concept for neurosteroids as potential therapeutics in
neurodegenerative
diseases
(27).
II.
NONVIRAL DELIVERY SYSTEMS TO
DELIVER AGENTS TO THE NERVOUS SYSTEM
Although the expression of specific proteins by transfection with viral vectors has been a commonly used technique, this method of drug delivery has certain disadvantages (28-30). A number of nonviral approaches to drug
delivery to the nervous system have been developed, including (i) intraventricular infusion of neuroactive agents,
(ii) injection or implantation of polymeric systems, (iii)
implantation of genetically engineered cells or stem cells,
and (iv) use of liposomes. These approaches are summarized in the following sections.
A. Intraventricular Infusion of
Neuroactive Agents
Poorly soluble agents and unstable peptides are often administered into the lateral ventricles either as single injections or via permanently installed cannulae (31,32). The
advantages of these approaches are that the dosage and rate of
drug administration can be controlled, and the results
resemble a slow intravenous infusion if the drug is readily
distributed into the peripheral bloodstream. However, intracerebroventricular (ICV) injection of drug results in distribution to the ependymal surface of only the ipsilateral
brain because of the unidirectional flow of cerebrospinal
fluid within the brain. The major disadvantage of ICV drug
administration is its invasiveness and the possibility of infection at the site of penetration of the BBB.
B. Injectable and Implantable Polymeric Systems
as Drug Carriers
1. Drug-Polymer Conjugates
Synthetic polymer materials have been used as drug carriers
in several modalities (Fig. 1). Injectable drug-polymer
conjugates are produced by covalent binding of water-soluble polymers to a drug. The nature of the covalent bond
between the drug and the polymer should be such that the
bond is strong enough to be stable in the bloodstream but
easily cleaved once the conjugate has reached the target
site. This is often difficult to achieve. Moreover, only a
relatively small number of biologically active molecules
can be attached to the polymer molecule, thus requiring
relatively large amounts of drug-polymer conjugate to be
injected at the site of action. Approaches overcoming some of
these problems are discussed in the following sections.
2. Implants
Simple replacement therapy with polymeric implants of
nerve growth factor have been implemented in animal
1085
achieved in different ways. However, all of these simple
replacement approaches have three major limitations: (i)
site-specific delivery, (ii) the amount of drug that can be
administered by single administration, and (iii) susceptibility
of full-length peptides to enzymatic cleavage due to the
presence of various peptidases in the tissue. To solve some of
the stability problems, drugs can be incorporated into
biodegradable polymers, and an overview of these polymers is given in Chapter 5.
3. Osmotic Pumps
Figure 42.1 Some common approaches to administer neuroactive
drugs. 1. Drug covalently bound to the polymer. 2. Micro-spheres
(made of biodegradable polymers) containing neuroactive agents
can be injected either systemically, into the lateral ventricle, or
into the selected brain structure. 3. Microsponges can be
impregnated with neuroactive agent and administered locally. 4.
Osmotic pumps allow for steady release of neuroactive agent for a
prolonged time period (1-2 weeks). 5. Injections of neuroactive
agents directly into the lateral ventricle or parenchyma.
models of central cholinergic deficiencies (33,34) and of
peripheral nerve impairment in diabetes (24), in both humans and several animal species (35,36). Recently nerve
growth factor (NGF) was delivered locally by implantation of
a small polymer pellet providing slow release at a controlled
distance from the target site (37). The implants placed 12 mm away from the target cholinergic site were effective,
whereas the same implants placed 3 mm away from the
target site had no detectable effect. These findings strengthen
the notion that NGF delivery within a spatially restricted
area should be considered a desirable feature if the drug is
to be effective. Due to the larger size of the target areas in
the human brain than in rodent animal models, the concept
of pharmacotechtonics has been tested. This strategy
involves the creation of an array of local drug-releasing
loci to create large but spatially restricted and anatomically
defined fields of biological activity. Drug distribution can be
more controlled, and moreover this approach lends itself to
comparison with mathematical models (38). The geometry
and sites of implantations can be determined by
noninvasive diagnostic procedures, such as MRI, prior to
the surgical procedure. Local delivery, in conjunction with
pharmacokinetic modeling (39) and ste-reotaxic atlases
linked to MRI scanners, will eventually allow for
customized drug therapy for individual patients.
Replacement of other factors such as ciliary neurotrophic
factor (CNTF), lymphocyte inhibitory factor (LIF), and
brain derived neurotrophic factor (BDNF) has also been
Osmotic pumps are also often used in experimental animals. For instance, implantable pumps have been used in
primates to deliver dopamine or dopamine agonists
(40,41). The pump reservoir is typically installed subcutaneously, and a catheter links a cannula with the pump.
There are different sizes of pumps, suitable for small rodents or larger animals (commercially available "Alzet"
minipumps); the pumps are refillable, and newer models
allow for the adjustment of the delivery rates. The major
limitation of pumps is the possibility of a local immune
reaction at the site of delivery. In addition, due to the limited
diffusion of most peptidergic neurotrophic agents, the
majority of the agent is degraded before reaching the intended site of action.
4. Micro- and Nanoparticles
These delivery systems were reviewed previously
(4,42-48). Controlled release polymer systems not only
improve drug safety and efficacy but may also lead to new
therapeutics. Some of the frequently used polymers are
poly(sebacic acid-co-1,3-bis(p-carboxyphenoxy)propane),
poly(b-hydroxybutyrate-hydroxyvalerate), poly(lactide-coglycolide), poly(methyl methacrylate-acrylic acid),
poly(acrylamide-co-acrylic acid), and poly(fumaric-co-sebacic anhydride). Numerous micro- and nanoparticles
(some examples of which are shown in Fig. 2) have been
designed and tested in vitro and in vivo to demonstrate
superior effectiveness with concomitant reduction in neurotoxicity. Conventional oral or transdermal delivery is inadequate for the delivery of macromolecules such as proteins. Due to the short half-life of macromolecules such as
growth factors, micro- and nanocontainers made of different
polymers have been investigated as a means of their
controlled and prolonged release. Johnson et al. (49) developed biodegradable microspheres composed of lactic coglycolic acid polymer in which lyophilized macromolecules (human growth hormone) were complexed with zinc to
solve the problem of moisture-induced protein aggregation.
The system was tested in vitro and in a primate in vivo
model. A release of the protein for one month was
demonstrated, suggesting the possibility that such a system
Figure 42.2 Some examples of injectable nanoparticles as carriers
for neuroactive agents.
may be considered for chronic clinical use. Numerous
other nano- and microparticulate biodegradable and biocompatible delivery systems have been developed in the
last several years. For instance, rhodium (II) citrate, a recent member of promising antitumor agents, was complexed and encapsulated into poly (D,L-lactic-co-glycolic)
acid (PLGA) and poly(anhydride) microspheres (50).
Complexation in this case significantly increased the encapsulation efficiency and duration of release in both polymer
systems (50). However, problems that need to be dealt with
include the limited supply of the neuroactive agent, the
invasive aspects of micro- and nanoparticle administration,
and release kinetics that were not amenable to regulation by
physiological changes at the site or elsewhere. Two
interesting and novel approaches have been recently considered and tested: controlled release microchips and neurospheres. Briefly, in contrast to previous methods of controlling drug release from polymeric devices such as
pulsatile stimuli by an electric or magnetic field, exposure to
ultrasound, light, enzymes, changes in pH or in temperature,
new biotechnological approaches have led to the development of a solid-state silicon microchip that can provide controlled release of a single or multiple agents on
demand (51). Although it is too early to evaluate its usefulness for the delivery of neuroactive substances, it certainly seems promising. Neurospheres of multipotent and
restricted precursors may provide solutions for a longer
lasting and more physiological supply of biologically active compounds, either singly or in combination (52-54).
5. Liposomes
Cationic liposomes may have a significant potential for
clinical applications in gene therapy for the disordered central
nervous system (CNS) (55). Recently it has been reported
that intracerebroventricular or intrathecal injection of
cationic liposome-DNA complexes can produce
significant levels of expression of biologically and
therapeuti-cally relevant genes within the CNS such as
nerve growth factor (NGF), granulocyte colonystimulating factor (G-CSF), and choline acetyltransferase
(ChAT) (56). Technical aspects to achieve maximal gene
transfer into brain cells using a plasmid DNA-cationic
liposome complex have been discussed by Imaoka et al.
(57). These authors have administered plasmid DNAcationic
liposome
(lipo-polyamine
of
dioctadecylamidoglycyl spermine) complex to 3-6 months
old male rats using an osmotic pump. They report an
increase of approximately up to two orders of magnitude
in transfection efficiency compared to one obtained by a
single injection. The authors propose that the continuous
injection approach may be safe and effective in increasing
the transfection efficiency. Another group led by Yokota (58)
examined the effects of a calcium-dependent cysteine
protease (calpain) inhibitor entrapped in liposomes in
delaying neuronal death in gerbil hippocampal CA1
neurons following a transient forebrain ischemia. Selective
neuronal damage induced by forebrain ischemia in the CA1
region of the hippocampus, and calpain-induced proteolysis
of neuronal cytoskeleton, were prevented by
administration of the inhibitor in a dose-dependent manner
(58). Evaluation of transfection efficacy of a plasmid vector
complexed with three different cationic liposomes into two
experimental rodent and human malignant glioma cell lines
and the mouse 3T3 fibroblast were studied by Bell et al.
(59). The transfection efficacy and cytotoxicity of the
liposomes were reported to vary quantitatively and qualitatively between cell lines. These authors suggest that their
results support a potential application of cationic liposomes in both experimental and human malignant glioma
gene transduction. Further studies on liposomal transfection
of normal and neoplastic cells derived from the CNS will
likely be very useful in helping to ascertain the particular
merits of liposome-mediated gene transfection (59).
Although the emphasis has been on utilizing liposomes in
gene delivery to the CNS, this by no means limits their
use to gene transfection (60-63).
C. Therapeutic Approaches Employing Cells
1. Genetically Engineered Cells
In order to provide longer term neurotrophin delivery without
the need to refill the containers or reduce the frequency of
reimplantation of delivery devices, several groups
(5,64-66) have developed implantable polymeric devices
containing genetically engineered cells that can produce,
for example, a missing trophic factor (Fig. 3). This strategy
has been tested in animal models, including primates (67).
Either primary cultures or genetically engineered cells producing a missing factor can serve as "long term effective
mini-factories," and various cell types used for these pur-
1087
Figure 42.3 Genetically engineered cells and stem cells. Different
cell lines, primary cell cultures, and genetically engineered cells
producing a neuroactive agent can be directly injected into the
brain as a cell suspension, or prior to administration cells can be
microencapsulated in biocompatible polymers. Neural stem cells
with the capacity to renew and produce the major cell types of the
brain can be used for cell replacement therapy in neurological
disorders. (See Section C.3, Stem Cells.)
poses have been reported and reviewed, including pheochromocytoma cells (PC 12) (68), fibroblasts, and NIH
3T3 cells genetically altered to produce growth factors
(66,69,70). Although fully mature primary cultures or genetically engineered proliferating cells of nonneuronal origin
can replace missing peptides, they are either (i) deliberately
physically separated from the environment at the
implantation site to prevent tumor formation (e.g., by encapsulation or by placement of cells within a retractable
implantation device) or (ii) in contact with the immediate
microenvironment, their phenotype not allowing them to
integrate and make functional connections (e.g., PC 12
cells, fibroblasts).
3. Stem Cells
Replacement strategies using stem cells have recently become an attractive way to overcome the problems of cell
integration and of acquisition of normal brain functions
(71). Adult CNS stem cells can replace neurons and glia in
the adult brain and spinal cord (72) and can also give rise to
other cell types such as skin melanocytes and a range of
mesenchymal cells in the head and neck (73). Stem cells
may integrate appropriately into both the developing and
the degenerating central nervous system and may be
uniquely responsive to some types of neurodegenera-tive
conditions (74). Neural-derived stem cells are selfrenewing under the influence of mitotic agents such as
fibroblast growth factor (75), epidermal growth factor
(76,77), BDNF (78), and other factors (71,79-82). These
cells can differentiate into either neuronal or glial cells and
therefore can be used to replace neurons that are damaged or
destroyed in defined neuronal structures, such as dopaminergic nigral neurons in Parkinson's disease, or hippo-
campal neurons (70,76,83-87). Neural stem cells cultured
from human embryos can be grown for extended periods of
time while retaining the capacity for neuronal and glial
differentiation. The ability to generate human neural tissue in
vitro allows for screening of neuroactive compounds and
provides a source of tissue for testing cellular and genetic
therapies for CNS disorders (88). Neurospheres of
multipotent and restricted precursors may provide solutions for a longer lasting and more physiological supply of
missing biologically active compounds, single or multiple,
(52,54,89). Most importantly, stem cells have the advantage
of establishing functional connections within the nervous
system, a property that cannot be achieved with any
polymeric drug delivery system, at least not in cases when a
large proportion of neurons is lost. Accounts of the current
status of stem cells and their biology and potential in
treating neurological disorders are available in recent
reviews (70,87,90). Obviously, ethical issues are of importance in implementing stem-cell strategies (84,86).
III. BLOCK COPOLYMER MICELLES
AND VESICLES
Block copolymer micelles (Fig. 4) have a great potential
as delivery systems for the administration of neuroactive
agents. Previous work (91,92) provided some seminal information in this regard, but much fundamental work relating to physical, morphological, and biological (pharmacological) properties must be done before block copolymer
micelles and vesicles can be used either as diagnostics or as
therapeutics. Thus far, only spherical micelles have been
studied from the physicochemical and biological aspect.
Other morphologies were only recently produced and identified using EM (93-95) and some sporadic in vivo studies
have been reported (cylinder shapes delivered to lungs)
without physical-chemical characterization. Our group has
been involved in fundamental studies addressing the questions of interrelationships between morphological features
(shape, size) and physiochemical properties of tailor-made
micelles containing either fluorescent labels (96,97),
highly lipophilic radiolabeled agents such as benzopyrene, or
poorly soluble bioactive agents such as dihydrotestoster-one
and FK506 (96,98,99). A recent overview of the physical
properties of block copolymer micelles used in vitro and
in vivo in studies by several groups, including ours, is
available in (92) and (98).
A. Biodegradable Block Copolymers for
Development of Micellar Delivery Systems
One of the promising biodegradable and biocompatible
polymers for micellar delivery systems is polycaprolac-
Figure 42.4 Representative types of block copolymer micellar
delivery systems. 1. Corona forming block has attached ligand
to provide site-specific delivery of neuroactive agents. 2. Inverse
micelle for the delivery of hydrophilic neuroactive agents. 3.
Block copolymer micelle for the delivery of lipophilic neuroactive
agents. 4. Block ionomer micelle suitable for the delivery of
antisense oligonucleotides and DNA.
tone-fc-poly(ethylene oxide) (98). The individual components, polycaprolactone and polyethylene oxide, were explored previously for a variety of biomedical applications
(110,111).A list of some core forming polymers is given in
Table 1. Polycaprolactone, the hydrophobic core-forming
block of the micelles, is a biodegradable polymer used as
(i) a structural material in the production of medical devices such as implants, sutures, stents, and prosthetics, (ii) a
carrier for a variety of drugs (112), (iii) in paste form for
drugs (113,114), and (iv) a nanoparticulate ocular delivery
system (115). Polyethylene oxide, the hydrophilic shellforming block of the micelles, imparts blood compatibility to
material surfaces (116) and is commonly used in micellar
drug delivery systems (91). This polymer lends itself to
chemical modifications that can enhance site-directed
delivery. Micellar delivery systems for neuroactive agents
are described in the following paragraph.
B. Micelles for the Delivery of
Neuroactive Agents
Several types of micelles formed from different polymers
and copolymers have been developed (Table 2), some of
which could be useful for the delivery of neuroactive
agents. Several considerations arise when considering
polymer micelles as drug carriers for CNS-based therapeutics. For instance, a high partitioning of the drug into the
micelle is required because otherwise one would have to
administer large amounts of micelles. Similarly, the poly-
mer must be biodegradable, biocompatible, and with sufficiently low critical micellar concentration (CMC) to
achieve a longer length of time in the bloodstream to allow
the drug to reach its site of action. Modifications to optimize polymer-drug interactions and high stability of micelles in the blood have been recently reviewed (127). In
order to enhance endocytosis and ultimately transcytosis of
micelles containing neuroleptic agents across the BBB,
ligands were attached to the polymer micelles. An improved internalization of derivatized pluronic micelles has
been demonstrated using primary cultures of brain microvessel endothelial cells (BBMEC) (128-130). Nevertheless, micelle carriers for drug delivery to the brain still
have some limitations:
1. Brain endothelial cells take up a relatively small
amount of micelles designed so far. Consequently, the
amount of drug delivered is relatively small. However, if
the drug has a high potency, it should be still possible to
obtain the desired biological effects.
2. Drugs to be incorporated into the micelles should
have very low capacity to cross the BBB. This is not the
case with highly lipophilic agents, but many peptides and
oligonucleotides are good candidates for micellar delivery
systems to the brain.
In addition to the micelle-based strategies outlined here,
there is another strategy that has been explored to a limited
extent, namely the exploitation of specific interactions of
the polymers themselves with the membrane and membrane transporter proteins found in the brain microvessel
endothelial cells that form the BBB. In this case it is not
necessary to have micelles, since the monomer itself can
modify the BBB permeability. Two proteins within the
BBB are targets of such an approach: P-glycoprotein (Pgp) and multidrug resistance-associated protein (MRP)
(128,129). Modifications of P-gp and MRP and possibly
other transporters in the BBB with polymeric formulations
may considerably facilitate the accumulation of neuroactive
agents within the brain. Among the polymers tested so far,
pluronic polymers seem to be particularly effective in
inhibiting P-gp drug efflux in the brain microvessel endothelial cells (106). Some drugs can also change the permeability of the BBB, such as cyclosporin A. Unfortunately, cyclosporin A in equivalent concentrations to
Pluronic P85 seriously disrupts the integrity of the brain
microvessel endothelial cells (127). Some examples of
drugs with effects on the nervous system and of their
delivery vehicles are given in Table 3. Polymers as trans-
port-modifying agents have both advantages and limitations:
1.
Polymers exhibit relative selectivity. However,
since P-gp and MRP are also present in liver,
the drug could accumulate in this organ, affecting
normal functions, in particular metabolic processes.
2. Less damage occurs to the microvessel endothelial
cell integrity.
3. Some polymers are effective below the CMC.
C. Micelles for the Delivery of Antisense
Oligonucleotides
1. Problems in the Delivery of Antisense
Oligonucleotides
The use of antisense Oligonucleotides to control protein
expression has received considerable attention due to their
relative ease of synthesis and specific use. Commonly,
short (between 15 and 20 bases) chemically modified nucleic acids are employed. They hybridize to complementary nucleotide sequences in accessible regions of mRNA
molecules, thus blocking expression of the encoded proteins. Two major problems are associated with the use of
antisense Oligonucleotides: (i) the identification of accessible
regions in mRNA to which the Oligonucleotides can
hybridize, and (ii) the delivery of the Oligonucleotides into
the cell and to their target (136). The accessibility of an
mRNA sequence is determined by the primary nucleotide
sequence, its three-dimensional structure, and the presence of
associated proteins. This combination of factors has
made the design of effective antisense Oligonucleotides a
difficult task, and experiments involving the use of poorly
designed antisense Oligonucleotides often produce misleading results. However, once a candidate oligonucleotide
has been synthesized, several challenges arise in delivering it
effectively to its target. A first consideration is the presence
of various endo- and exonucleases in serum that can
degrade the Oligonucleotides and destroy their biological
activity (137). Also, some types of antisense Oligonucleotides (e.g., phosphothiorate) are highly charged
polyanions and bind extensively to serum proteins (138140). Such oligonucleotides have a very limited ability to
cross cellular membranes, and this reduces the amount of
oligonucleotides that can reach their nuclear target (141,
142). Furthermore, the binding of serum proteins by
oligonucleotides may also alter the protein activity, which
may be misinterpreted as an effect of the oligonucleotides
on nuclear targets. A second consideration is that the
amount of oligonucleotides taken up by different tissues
and cells varies considerably. Studies in rodents have
shown that the majority of intravenously injected oligonucleotides distribute to the kidney and liver, where they may
be degraded and lose biological activity (143-144). Also,
these studies showed that the biodistribution of oligonucleotides is generally much greater in the intestine,
bone marrow, skeletal muscle, and skin than in the brain.
This presents additional concerns for the delivery of
oligonucleotides to targets in the central nervous system.
The actual mechanisms by which antisense oligonucleotides enter into cells are currently under debate, but they
are thought to involve fluid-phase pinocytosis and/or receptor-mediated endocytosis (145). A third concern is that
once the oligonucleotides have successfully entered into a
cell, they are subject to degradation. Confocal and electron
microscopy studies have shown that the majority of internalized antisense oligonucleotides enter into the cellular
endosomal and lysosomal systems (146). These compartments may have acidic pH and contain enzymes that degrade the oligonucleotides and destroy their biological
activity. It is clear, however, that a portion of the administered oligonucleotides either escapes from the endosomal/
lysosomal systems or bypasses them altogether and enters
the cytoplasm to diffuse into the nucleus, by poorly understood mechanisms (141). The cytosolic environment also
contains a variety of exo- and endonucleases and proteins,
which presents problems similar to those encountered in
the serum. An additional concern arising from microscopy
studies is the existence of an efflux of oligonucleotides
from the nucleus, likely through a passive diffusion mechanism via nuclear pores (146), and from the cell (147-149).
These phenomena must also be considered when targeting
antisense oligonucleotides to the nucleus.
2. Early Approaches in Antisense Oligonucleotide
Delivery Systems
Short-term antisense therapy is often marked by the development of two common toxicities, namely activation of
the complement system and increase in blood clotting time
(150,151). However, clinical studies have shown that these
effects are minor due to the relatively short half-life (30-60
minutes) of oligonucleotides in the serum, which may be
due to binding of serum proteins to oligonucleotides
(145). As well, chronic administration of antisense oligonucleotides in rodent models leads to the induction of immune responses, characterized by lymphoid hyperplasia,
splenomegaly, and monocyte recruitment to a number of
tissues (151). In consequence, there has been considerable
interest in developing delivery methods that can minimize
these degradative and immune drawbacks to the use of oligonucleotides. Early approaches focused on improving the
cellular uptake of antisense oligonucleotides by coupling
them to polycations such as polylysine (152) and polyethylenime (153), or, more successfully, to polycationic lipids/
lipid formulations (154,155). Other approaches involved
targeting antisense oligonucleotides to cell surface receptors
such as the folate receptor (156), transferrin receptor (157),
and asialoglycoprotein receptor (158), to stimulate receptormediated endocytosis. Another strategy involved the
coupling of oligonucleotides to typical membrane entities such as cholesterol (159) or to fusogenic peptides
(160). These in vitro uptake enhancers have been successful
in tissue culture systems, with significant increases in the
uptake and nuclear localization of the oligonucleotides.
However, it is noteworthy that the internalization profiles of
oligonucleotides in vitro often differ considerably from
their behavior in vivo (145). Furthermore, these early approaches did not focus upon minimizing the immunogenic-ity
and instability of the oligonucleotides, which has been
addressed by the micellar delivery approach described
below.
3. Delivery of Antisense Oligonucleotides
Employing Copolymers
Although some oligonucleotides are relatively stable following their administration by a variety of routes (intravenous, intraperitoneal, subcutaneous, intracerebroventricular) (161), delivery of antisense oligonucleotides by
delivery systems has been proposed in order to reduce the
immunogenicity and protect them from the physiological
degradative processes referred to above. The major work
in this area has been carried out by Kabanov's group, using,
first, hydrophilic polymeric vesicles (162), and subsequently,
polyion complex micelles with a protein modified corona
(163). A hydrophilic polymer, Nanogel, was used to create
vesicles with an average particle size of 120 nanometers
(162). This polymer is formed from cross-linked
polyethyleneimine (PEI) and carbonyldiimidazole-activated poly(ethylene glycol) (PEG), thus building on the
PEI-coupling approach of Boussif (153), which focused
on enhancement of cellular uptake. Nanogel particles have
been employed for the delivery of antisense phosphothiorate oligonucleotides (SODN), targeting the mRNA of the
human multidrug resistance gene, mdrl (162). These studies
have shown that the Nanogel vesicular approach attains a
greater cellular localization of SODN in a human carcinoma cell line in comparison to the administration of free
SODN. The effectiveness of the Nanogel-incorporated
SODN was demonstrated by its ability to inhibit the expression of P-glycoprotein, a major cellular pump involved
in the efflux of cytosolic drugs.
A significant reduction in delivery vehicle size was
achieved by Kabanov's group with the synthesis of polyion
complex micelles incorporating SODN (163). These micelles contain poly(ethylene glycol)-PEI graft copolymer
complexes that self-assemble with SODN to form 40 nanometer particles, each consisting of a PEI/SODN neutralized core surrounded by a poly(ethylene glycol) corona.
Transferrin molecules attached to the poly(ethylene glycol) corona create polyion complex micelles (75 to 103
nanometers) facilitating the internalization of SODN into
cells. These studies (163) have indicated that SODN incorporated in polyion micelles with a protein modified corona
have a significantly greater ability to inhibit the expression
of the P-glycoprotein drug efflux transporter in several
cancer cell lines.
4. Micelles for the Delivery of DNA
Some of the most frequently applied techniques to
transfect cells are (i) precipitation with calcium phosphate
(164-170), (ii) polybrene (171), (iii) electroporation (172),
(iv) microinjection (173), (v) modified viral vectors
(174,175), (vi) microspheres (47), (vii) liposomes (176178), and (viii) polycation delivery systems (121,179181). There are several commonly used protocols for cell
transfections, the most frequently used being precipitation
with calcium phosphate (164,182).
Kabanov's group has contributed greatly to the delivery
systems of genetic materials. The methodology is based
on formation of soluble interpolyelectrolyte complexes
(IPECs). The term IPEC in polymer science relates to the
products of reaction of oppositely charged polyions, and in
the case referred to here polycation-DNA complexes
represent a special kind of IPECs relevant to biological
issues. A critical assessment of transfection approaches in
mammalian cells using DNA-IPECs and more common
methods such as calcium phosphate precipitation and lipofectin is given in several reviews, e.g., Ref. 179. Polycations as building blocks for DNA complexes can be
relatively easily conjugated with ligands and undergo receptor-mediated endocytosis (179). Among the most frequently attached ligands are asialoglycoprotein, insulin,
and transferrin. Asialoglycoprotein receptors play a critical
role in hepatocytes and allow for targeted delivery of DNA
to the liver (183,184). Insulin and transferrin receptors are
present in many cell types and provide an endocytotic internalization of the delivery system (insulin receptor) or
vesicular transport followed by the return to the cell surface (185). Antibody molecules have also been linked to
IPECs to achieve cell and tissue specificity (186). Since
neural cells express a wide variety of receptors, some of
them specific for (sub)classes of neurons (e.g., cholinergic,
dopaminergic, gabaergic), IPECs linked to ligands recognizing these receptors offer an attractive approach in drug
and gene delivery to the nervous system.
calized (site-specific) physiologically controlled drug release is always desirable. These goals are being gradually
achieved by the increasing availability of new functionalized biopolymers, and "smart polymers."
Smart polymers are hydrogels that undergo fast, reversible
changes in microstructure from a hydrophilic to a hydrophobic state (187). Triggers that can produce these
changes include neutralization of charged groups by a shift
in pH, the addition of an oppositely charged polymer, a
change in temperature or ionic strength, or the formation
of interpenetrating polymer networks (187). Stimulus-responsive or smart polymers have been used mainly for bioseparations but also for the development of drug delivery
systems. One of the models based on smart polymers is a
glucose-responsive insulin loaded polymer matrix (188).
The number of drug delivery systems utilizing smart polymers is very limited, and much work remains to be done in
the area of their synthesis and structure-property relationship studies before they can be considered for clinical
applications.
In addition to drug release from polymeric devices such
as pulsatile stimuli by an electric or magnetic field, exposure
to ultrasound, light, enzyme, or change in pH or temperature,
new biotechnological approaches have led to the
development of solid-state silicon microchips that can provide controlled release of single or multiple agents on demand (51). Although it is too early to say if microchips
will supplement or replace classical delivery systems, they
represent a significant step forward in biotechnological approaches to administer neuroactive agents in a more controlled manner.
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