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Drug delivery to the nervous

system
Dusica Maysinger and Anne Morinville

Delivery of drugs to the nervous system remains a challenge despite advances

in our understanding of the mechanisms involved in the development of
neurodegenerative disorders and the actions of neuroactive agents. Drug
accessibility to the central nervous system is limited by the blood-brain barrier;
although the peripheral nervous system is more accessible than the central nervous
system, problems are still encountered, mainly owing to the poor stability and
considerable side effects of many neuroactive compounds when administered
systemically. Microencapsulation of neuroactive compounds and living cells
producing such substances can overcome some of these shortcomings for delivery
to the nervous system.

In the treatment of diseases or conditions that result Drug-delivery systems

from the lack of simple hormones or peptides, the ‘Microcapsules’ contain an agent in their centre sur-
administration of these compounds in a controlled rounded by a polymer or a macromolecular carrier. A
fashion could provide therapy. Conditions such as dia- drug (or other active agent, or a living cell) is in the
betic neuropathy, amyotrophic lateral sclerosis (ALS) core of the microcapsule and the surrounding poly-
and Huntington’s disease, and possibly also Alzheimer’s mer layer can vary in thickness and permeability, and
and Parkinson’s diseases, could benefit from this type their diameter usually ranges between 1 and 2000
of approach. Nevertheless, although some of the can- micrometresi. Microcapsules are also known as
didate compounds for treatment have been identified, microspherules, spansules, and coate’d granules, pellets
an adequate means of long-term delivery to the ner- or seeds’, and can have several separate cores
vous system is still lacking. Only a limited number surrounded by a polymer or a mixture of polymers.
of small, stable molecules acting on neurons or non- ‘Microparticles’ are matrices containing drugs (or
neuronal tissue are available for long-term treatment other agents) more or less homogeneously distributed
that can be administered orally. However, peptide drugs (suspended) throughout the entire matrix with no
and small peptides are easily degraded by proteolytic distinct core or envelope. Their dimensions are in the
enzymes and hence cannot reach their site of action micrometre to nanometre range. ‘Microspheres’
when administered in this way. The strategy for the (nanospheres) represent examples of microparticles
administration of these readily hydrolysable molecules (nanoparticles).
for the treatment of neurodegenerative conditions of Examples of microparticles and microcapsules and
the central and peripheral nervous system (CNS and other delivery devices containing neuroactive agents
PNS, respectively) can assume two directions - either or living cells for administration to tlhe CNS and PNS
the defective or absent gene is replaced, with the are provided in Table 1.
expectation that the missing compound will then be
produced, or the missing compound is provided Targeted drug delivery and the related problems
directly. This article focuses on the latter approach, Two main categories of targeting exist - passive and
which can be achieved by direct delivery of the mol- active. Passive targeting occurs because of the body’s
ecule or through the use of living cells that produce natural responses to foreign material; macrophages
the missing substance. from the liver and the spleen play the pivotal role in
these actions. Small particles are not as subject to retic-
uloendothelial-system (RES) uptake as larger ones,
D. Maysinger [dmaysing@pharma.mcgill.ca) and A. Morinville are at
and polymeric materials can also influence this process
the Department $Pharmacology and 7’herapeutics, McGill University,
3655 Drummond Street, Room 13 14, Montreal, Quebec, Canada - for example, hydrophilic, neutral surfaces are
H3G lY6. favoured over more lipophilic larger particles. In

TIBTECH OCTOBER 1997 (VOL 15) Copyright 0 1997, Elsevier Scmce Ltd. All rights reserved. 0167 - 7799/97/$17.00. PII: SO167-7799(97)01095-O
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contrast, active targeting may involve the attachment

of a receptor device or vector molecule to the drug- Table 1. Delivery of drugs and cells to the central and
delivery system. If the target site is the liver or spleen, peripheral nervous systems
the drug-delivery vehicle should be designed to acti-
vate the complement system in order to promote Drug-delivery devices
RES uptake. If the target is another organ, the vehicle
must avoid RES uptake and should include a vector Microcapsules Gelforms
molecule that will direct it to this organ. However, this Microsphere-matrix
type Collagengels
Liposomes Osmoticmini-pumps
task remains difficult and only limited success has been Micelles Adhesivepatches
achieved so far. Conjugates

Liposomes Living-cell-delivery devices

Since their discovery by A. D. Bangham in the
196Os’, liposomes have received much attention as Encapsulatedcells Hollowfibres
drug carriers. Conventional liposomes resemble Injectionof dispersedcells Collagengels
plasma membranes, consisting of phospholipid mol-
ecules with a polar head with two hydrophobic tails,
forming a bilayerj. In contrast, micellar subunits pos-
sess one such tail and form a single layer3. Liposomes research into the long-term administration of drugs to
are biocompatible, nontoxic and biodegradable2J,“, the CNS has classically been directed at permanently
and offer the possibility of carrying hydrophobic, installed devices. In order to avoid inconvenient inse:r-
hydrophilic or amphiphilic molecules3. Despite these tions and the possible removal of large implants or per-
numerous attributes, liposomes have not gained wide- manently installed stainless-steel cannulae, several types
spread use as drug-delivery systems, due to their insta- of injectable drug formulations, including microcap
bility in viv$J,“. Classical liposome preparations, steric- sules and microparticles, have been applied in neuro-
ally stabilized liposomes and immunoliposomes are all scientific research, showing some promise for clinical
susceptible to clearance by the immune system’J,j. use. On the other hand, systemic or local drug deliv-
Most work on liposomal applications has centred on ery is needed for administration to the PNS, for exam-
the delivery of anticancer agents and has culminated ple in diabetic neuropathy. Regardless of the ultimate
in the liposomal formulation of doxorubicin3.5. goal, one of the major obstacles remains the choice of
Recent advances in liposomal formulations include construction materials; the biocompatibility and con-
cationic liposomes used to entrap various polynu- trolled rate of drug release of the material are impo:r-
cleotide@. In addition, cationic liposomes have been tant variables in the selection process. Table 2 lists some
complexed with DNA; these are synthetically based examples of materials used for the encapsulation of
nonviral carriers of DNA vectors for gene therapy”, neuroactive agents.
but the in vim lifetime of these promising formulations Polymers that can be used for the preparation of
remains to be determined. At present, the working in implants or microspheres may be either biodegradable
vivo range of liposomes is restricted to the nanometre or nonbiodegradable, with both types having eno-r-
scale2 as long-term circulation depends on size: the mous potential for drug delivery in the CNS and
larger the liposome, the faster the clearance. Because for use as vehicles for transplanting viable cells7.8.
the action of neuroactive agents (for example, neuro- Polysulfones’, poly(acrylonitrile-co-vinyl chloride)*O,
trophic factors) is mostly achieved by their coupling ethylene-vinyl acetate (EVA) and hydroxyethyl-
to a receptor on the plasma membrane, causing an methacrylate-methyl-methacrylate copolymer
intracellular cascade of events, the formulation of these (HEMA-MMA)7,8 are frequently used nonbiodegratl-
molecules for delivery must allow sustained extracel- able materials. These water-insoluble systems yield
lular release. Sterically stabilized liposomes, which are capsules that are mechanically and chemically stable
taken up by endocytosiss, are therefore unsuitable for and are convenient for cell implantation’. The pol;r-
this purpose. Finally, owing to the often potent nature mer acts as a semipermeable membrane, allowing the
of neuroactive agents, sustained drug release, entailing diffusion of the neuroactive agent. However, encapsu-
extended in vim stability of the delivery system, lation of living cells using these types of materials pn-
remains difficult with liposomes. In conclusion, lipo- sents numerous difficulties because of toxic effects of
somes are presently unsuitable for the long-term solvents on cells, either during the formation of ther-
administration of drugs to the CNS and PNS unless moplastic-based capsules or because of residual solvent.
adequately stabilized. Biodegradable polymers can be either synthetic or
natural and may be degraded in vivo both enzymati-
Microcapsules and microparticles tally and nonenzymatically. Their byproducts should
A delivery device for long-term treatment of the be biocompatible, nontoxic and readily excreted. Only
nervous system needs to possess a lifetime that exceeds a few immunologically tolerable biodegradable poly-
that of the entity it is carrying, which may be, for mers are available. Human serum albumin and bovine
example, a peptide molecule or a living cell. Due to serum albumin have been extensively used for target-’
the difficulties in penetrating the blood-brain barrier, ing anticancer drugs, insulin, and monosialoganglioside

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Table 2. Examples of materials used for encapsulation7.9JOJ3

Name Source Biodegradability Comments

Humanserumalbumin, Natural Biodegradable Nontoxic, immunologically

bovineserumalbumin tolerable
Collagen,haemoglobin, Natural Biodegradable Nontoxic, immunologically
gelatine,chitosan,alginate tolerable,doubtfulpurity
andpoly-L-lysine
PolyOactide-co-glycolide) Synthetic Biodegradable Versatile,controlleddrug release,
erodable
Polyhydroxyalkanoate Naturalor synthetic Biodegradable Thermoplastic,widevariety of
repeatingsubunits,controlled
drugrelease
Ethyl-vinylacetate, Synthetic Nonbiodegradable Thermoplastic
hydroxyethyl-methacrylate-
methyl-methacrylate
copolymer
Polysulfones, Synthetic Nonbiodegradable Retrievalafter implantation
polyfacrylonitrile-co-vinyl
chloride)
Fumaricandsebacicacids Synthetic Biodegradable Adhesivemolecules,increased
in viva lifetime,usefulfor drug
deliveryto gastrointestinaltract

(GMl). Other natural biopolymers, such as collagen, ester backbone, with numerous va:riations in the R-
haemoglobin, gelatine, chitosan, alginate and poly-L- pendant chain’*. Most poly-R-(-)-Shydroxyalkanoic
lysine, have been employed as carriers for parenteral acids are composed of a three-carbon-atom backbone
administration’t.la.13, but their cost and lack of purity and up to eleven carbon atoms in the pendant chainIs.
have hindered their use. Therefore, several types of The structure for these poly-R-(-)-3-hydroxyalkanoic
synthetic biodegradable polymers have recently been acidslsJ” is [-0-CHR-CH,-CO-O-],, where R rep-
developed and used for microencapsulation, because resents the pendant chain. This variation in repeat-
their processing conditions, availability and cost can be ing units confers a greater flexibility for tailoring the
controlled more efficiently’. agent-release properties compared to PLGAs. Be-
Among the biodegradable polyesters, which hold cause they are part of the natural biosynthesis-
considerable promise for the controlled delivery biodegradation cycle, PHAs have few or no side
of proteins, are those based on poly(lactic acid) effects and, under aerobic conditions, they degrade to
and poly(glycolic acid). Poly(lactide-co-glycolide) water and carbon dioxide’“. The most widely used of
(PLGA), commonly used in sutures, provides great these thermoplastic esters is poly(3-hydroxybutyrate),
flexibility in terms of composition, thus allowing tai- commonly abbreviated to P(3-HB) or PHB. PHB is
lored drug release. The biodegradation and tissue reac- often employed in conjunction with hydroxyvalerate
tions of PLGA have been extensively investigatedr”,ls. to form copolymers such as poly(3hydroxybutyrate)-
PLGA is a linear polyester prepared by ring-opening (3-hydroxyvalerate), P(3HB-3HV). PHAs have
polymerization and subsequently purified to yield a been employed to microencapsulate, among other
polymeric powder with the chemical structure things, anticancer drugs such as l-(2 chloroethyl)-3-
[-H2C-CO-O-],, [-CH(CH3)-CO-O-],,, and it cyclohexylnitrosourea (CCNU)20 and 2’,3’-diacyl-5-
hydrolyses by an acid- or base-catalysed reaction to fluoro-2’-deoxyuridine.
form lactic and glycolic acids. The degradation prop-
erties of PLGA depend on the molar ratios of the two Block-copolymer micelles in drug delivery
monomers in the polymer chain, the molecular mass, Traditionally, micellar drug-delivery systems have
crystallinity, size and shape of the device, and on the been formed from small-molecule surfactants and
implantation site 16. For example, the time taken for amphiphilic block-copolymer molecules. The incor-
PLGA copolymer to biodegrade can be varied from poration of the drug into small-molecule surfactant
about 30 days to almost a year1ss17 by adjusting the micelles is quite low, representing one of their major
lactic acid:glycolic acid ratio. limitations as successful delivery systems. In contrast,
Polyhydroxyalkanoates (PHA) represent another block-copolymer micelles can incorporate agents to a
class of biodegradable materials amenable for micro- greater degree. Block-copolymer micelles are formed
encapsulation and are potentially superior to PLGA. from individual block-copolymer chains containing a
These biodegradable plastics are synthesized by a num- hydrophobic block and a hydrophihc block. A variety
ber of bacterial species’s,‘“. The majority of PHAs of shapes and sizes (between 10 and 100 nm) can be
contain from three to six carbon atoms in the poly- produced using technologies developed by Eisenberg

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Figure 1
Block-copolymer micelles can be produced in a variety of shapes and sizes as illustrated in panels (a) to (d). (a) Small spherical aggregates
[PS(410)-bPAA13 without any additive]. (b) Large spheres (compound micelles from the block-copolymer 200-M). (c) Large compound
vesicles [aggregates from PS(410)-bPAA(13) with 253 FM HCI].(d) Univesicular aggregates [PS(410)-bPAA(13) with 90 pM CaCI,].

and co-workers2r~2a,23 (Fig. 1). These sizes are favour- agent. However, the decreased cytotoxicity was caused
able for avoiding capture by the RES. The amphi- by the slower release .of the covalently bound
philic character of the block copolymers enables self- adriamycine from the polymeric Carrie?.
assembly of nanometer-scale aggregates with a Recently, the focus has been on micellar systems
hydrophobic core and a hydrophilic outer shell in including noncovalently incorporated drugs, for
aqueous media. The hydrophobic core accommodates example micellar microcontainers. These types of
nonpolar drugs and the properties of the hydrophilic micelles are made of diblock and triblock copolymers,
shell can be adjusted to both maximize biocompatibil- and in aqueous solutions they form a water-soluble
ity and avoid RES uptake. shell (corona) with a micellar core formed &om the
Three main groups ofblock-copolymer delivery sys- hydrophobic loops of polymer chains. The dibl’ock
tems exist - polymeric drugs, micellar microcontain- and triblock copolymer micelles are both therrno-
ers and block-ionomer complexes21. Polymeric drugs dynamically and kinetically stable in aqueous media
are block-copolymer-drug conjugates in which the and are promising drug-delivery vehicles.
drug is covalently bound to the polymer carrier. Block-ionomer complexes are formed from
Common synthetic polymers and copolymers amphiphilic block copolymers with a charged
employed as carriers include poly(divinyl-ether-co- hydrophobic core-forming block. The compound to
maleic anhydride), poly(styrene-co-maleic anhydride) be incorporated into these micelles must have the
and homopolymers such as poly(L-lysine), poly(ethyl- opposite charge from the core-forming block. When
ene glycol) (PEG) and poly(L-aspartic acid). PEG- the block copolymer is mixed with a drug, a complex
polyaspartic-acid block copolymers were used to bind is formed because of electrostatic interactions between
adriamycine, and this system showed several advant- the core-forming block and the drug. This kind of
ages over adriamycine in non-micellar formulation. micellar block-ionomer complex has recently been
The major advantages were reduced binding to albu- shown to be useful for DNA complexing, allowing a
min, greater stability in vitro, lower systemic toxicity novel approach to DNA delivery consisting of the for-
and apparent higher effectiveness as an anticancer mation of DNA-block-ionomer complexes. Kabanov

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et al.*” demonstrated enhanced stability of 19-mer ‘Neurotrophins’ comprise a restricted group of neuro-
oligonucleotide incorporated into micelles of trophic factors that are structurally related to nerve
poly(ethylene oxide)-b-polyspermine in serum. Such growth factor (NGF).
types of ionomer-micelle complexes are potentially
useful as delivery systems in gene therapyad. The thera- Strategiesfor delivery
peutic potential of many drugs, particularly those that Various approaches have been developed to provide
are highly liposoluble, could be greatly enhanced by continuous delivery of neuroactive agents, and,
an adequate selection of block-copolymer micelles. although these have overcome some of the problems
The progress in polymer chemistry, in particular block of delivering these agents, numerous additional prob-
copolymers and rationally designed small peptides that lems remain. Even though drug delivery to the PNS
mimic the effects of large neurotrophic proteins, may appears simpler than delivery to the CNS, problems
offer new ways to deliver drugs to the PNS and CNS. such as the site of administration, linearity of release,
biocompatibility of the polymeric material and load-
Biologically adhesive molecules ing capacity have still to be solved. F’ermeabilizers of
An exciting new range of materials for microspheres the blood-brain barrier, osmotic pumps, slow-release
are the so-called biologically adhesive molecules. polymeric devices and particles, and igenetically engi-
These molecules possess advantages over the other neered cells represent some of the tested means of
drug-carrier systems, in that they exhibit increased neuroactive-agent delivery. In addit:lon, transfecting
residence time in vioo and, hence, the effects of the neural cells in vivu with viral vectors has been exten-
drug they carry could be sustained for longer inter- sively investigated and certainly deserves attention
vals. Traditional approaches to the development of (for reviews, see Refs 26-28). Table 3 summarizes
biologically adhesive molecules have centred on the some of the advantages and disadvantages of the main
use of hydrophilic polymers and hydrogels containing categories of nonviral delivery systems. The encap-
carboxyl groupsz5. Recently, a rapidly degrading sulation of growth factors and of cells engineered to
hydrophobic polymer consisting of polyanhydride secrete them is in its infancy; so far, only a limited
copolymers of fumaric and sebacic acids (FA:SA) number have been tested in in uivo models (rodents and
exhibited delayed passage through the gastrointestinal primates)y~ay-31. Several recently described examples of
tract when compared with other polymers due to an neurotrophin administration using microcapsules and
increase in biological adhesiona5. Poly(FA:SA) 20:80 microparticles will be summarized here, with an
nanospheres 4ct120 nm in size, fabricated using emphasis on those factors claimed to be effective in
phase-inversion nanoencapsulation, were detected in rescuing or preventing neurodegenerative conditions.
the cytoplasm of cells and also inside the Golgi appa- Essentially, two main approaches to the delivery of
ratus and secretory vesiclesas. Within the poly(FA:SA) neuroactive agents in encapsulated formulations exist
nanospheres, dicumarol, a low molecular weight com- - biodegradable polymeric microparticles and micro-
pound with low solubility and poor bioavailability, dis- capsules, and nonbiodegradable delivery devices such
played increased bioavailability in animals compared as hollow fibres that accommodate cells synthesizing
with other modes of oral administration’j. Similarly, trophic factors (see Table 1).
orally delivered insulin, encapsulated in a 50:50 blend
of poly(fumaric anhydride) and PLGA improved the Delivery of neuroactive agents using microcapsules and
regulation of glucose in rats in comparison with rats microparticles
fed an insulin in saline solution or a saline solution Among the most studied neurotrophins are NGF,
alone25. These results should, however, be interpreted brain-derived neurotrophic factor (BDNF), neuro-
with caution, as the animals were not diabetic and the trophins 3 and 4 (NT-3, NT-4) and glial-derived
usual parenteral administration of insulin was not neurotrophic factor (GDNF). The physiological roles
assessed in comparison with the oral nanosphere for- of these and related neurotrophic factors in nervous-
mulation. Systemic circulation of these bioadhesive system development and degeneration8v32.33, the sig-
molecules, required for the treatment of many condi- naling mechanisms related to them31 and the problems
tions, was not demonstrated. Despite this, these pre- of their delivery to the nervous system7,* have been
liminary studies offer exciting possibilities for the oral extensively reviewed.
delivery of a variety of compounds. The administration of growth factors such as NGE
insulin-like growth factor (IGF) or human growth
Delivery of neuroactive agents hormone (hGH) typically poses many problems,
‘Neuroactive agents’ is a broad term, and includes owing to limited oral bioavailability and short in uivo
several categories of peptide, protein and small mol- lifetimes3s. A number of human trials involving ciliary
ecules that act on the nervous system. Neurotrophic neurotrophic factor (CNTF) have yielded disappoint-
agents are substances capable of producing reparation, ing results36, often because of serious side effects
regeneration or protection of damaged cells in the ner- (toxicity)37; improvements in drug delivery could
vous system. ‘Neurotrophic factors’ are endogenously lower the therapeutic dose and hence reduce these
produced peptides that participate in the establish- side-effects36. Sustained-release formulations involv-
ment and maintenance of the neural phenotype ing microspheres composed of biocompatible
during embryonic development and adulthood. and biodegradable polymers could overcome these

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-
Table 3. Properties of common nonviral delivery systems for the peripheral and central nervous
systems*
-
Nonviral delivery Site of action (central Advantages Disadvantages
system or peripheral nervous
system)
-
Blood-brainbarrier Central Systemicuptake Highsystemicdoses,unlocalized
permeabilizers braindelivery
Osmoticpumps Centraland peripheral Cerebrospinal-fluid
delivery, Failurecould leadto potentiallytoxic
long-termdelivery dumping,refillingof pumps(canlead1
to infection),parenchymaldelivery
can causereactionat delivery site

Microspheresand Centraland peripheral Controlledsustainedrelease, Difficultretrieval after implantation,

nanospheres widechoiceof materials, implantscontainingneuroactive
potentialfor implantationof agentsneedreplacementfor
cells,stereotaxicimplantation, long-termdelivery
systemicdelivery

Genetically Centraland peripheral Long-termproductionof Rejection,therapy with

engineeredcells neuroactiveagent, controlled immunosuppressants, low availability
release of humancells,methodof delivery?
-

shortcomings, and also permit the use of high-toxicity Living cells for drug delivery
compounds. Recombinant human CNTF (rhCNTF), Many neurodegenerative conditions could pot’en-
which has possible applications in the treatment of tially be treated by the administration of neuroactive
ALS, was encapsulated in PLGA-and-chitosan micro- agents in a controlled manner through the use of liv-
spheres and also in dry alginate with chitosan using the ing cells, either wild-type or genetically engineered,
phase-evaporation method37. Sustained release was that secrete these molecules. However, immortalized
achieved in both cases, with a higher rate of release for cells grafted into the CNS often give rise to tum’our
the alginate-chitosan microspheres (2-l 2 days), formation in the host tissue’; the use of primary cells
whereas with PLGA-chitosan microspheres, drug is a possible strategy to overcome this problem.
release was observed up to 24 days37, but this formu- Nevertheless, cell transplantation has typically posed
lation has still to be tested if2 viuo. Colony stimulating problems due to the low availability of human cells
factor 1 (CSF-1) has been implicated in the differen- and immunorejection 41, leading to lifelong treatments
tiation, proliferation and survival of microglia, but with immunosuppressants. Immunoisolation offers
has typically posed delivery problems caused by enzy- the advantages of reducing immunorejection ;and
matic degradation38. Recombinant human CSF-1 introducing the possibility of xenograftsJ1. The
(rhCSF-1) was encapsulated in PLGA-chitosan immunoisolating device must allow the passage of
microspheres by the solvent-evaporation method3*. nutrients and oxygen as well as the active compound
These microspheres potentiated the microglial of interest, and must be biocompatible with both the
response to injury; the effect was slightly less when the host and the transplanted cell. Microencapsulated cells
microspheres were implanted in the peritoneum rather may thus become efficient biological pumps, over-
than at the site of lesion3R. coming some of the difficulties associated with the
Treatment of diabetic neuropathy could benefit from implantation of dispersed cells or repeated injections
the encapsulation of neuroactive agents. Reduction of ofdrugs (Table 3). PC12 pheochromocytoma cells .and
IGF activity has been shown in both human and non- fibroblasts are commonly used in these delivery sys-
human models of diabetesj9. The expression of IGF-I tems. Encapsulated cells have improved survival .and
is controlled by and mimics that of hGH, decreasing sometimes even produce more neurotrophin t:han
after the third decade in nondiabetic individuals3”13”. when grown as monolayer culturesZ9.30,37. Anchorage-
Recently, recombinant human growth hormone dependent cells such as fibroblasts need a support to
(rhGH) has been encapsulated in PLGA microspheres provide long-term survival, and matrigel, collagen and
using a cryogenic process35. In monkeys, injection of polyelectrolytes (alginate, chitosan) are commonly
these microspheres resulted in an increase of both used inside the polymer capsules.
rhGH and IGF-I for a period of one month3”, even The use of hollow fibres of nonbiodegradable poly-
though daily injections of rhGH failed to elicit the mers has the advantage of allowing the device to’ be
same response35. Therefore, growth factors within retracted when it is not needed or when the cells cease
microspheres represent an exciting combination to produce the drug, although surgery would be
for the potential treatment of neurodegenerative necessary. In some cases, the administration of neuro-
conditions of the PNS. trophins or neurotrophin mimetics”* in biodegradable

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microspheres is advantageous, because the polymer the lesion was reduced in animals with the hCNTF
will gradually erode, yielding biocompatible degra- cells’“. The retrieved implants exhibited a marked
dation products that can easily be eliminated by the decrease in the rate ofsecretion of CNTF with respect
body without the need for surgical removal. Techno- to the levels measured prior to implantation (12.7 ver-
logical approaches to and the current status of sus 45.1 ng dayi capsule-‘)I”. The reason for the
nonbiodegradable hollow-fibre encapsulation have decreased production was not determmed and the state
been extensively discussed in recent reviewssJ3. of the retrieved capsules was not asses:sed.Death of the
encapsulated cells would lead to decreased production;
Delivery using encapsulated living cells this could be caused by insufficient nutrient availabil-
A variety of devices have been developed for the irq an immune response to the cells or a reduction in
delivery of living cells, including microcapsules, cell viability due to the encapsulation conditions.
macrocapsules, h o 11ow fibres, tubular membranes, Nevertheless, the neuroprotective efIect on the corti-
intravascular devices and flat sandwich pouchesAl. The cal neurons innervating the striatum generated by
usual construction materials, such as HEMA-MMA these encapsulated hCNTF-producing cells provides
or poly-L-lysine with hardened polyanionic gel, encouraging results for the treatment of HD and other
are limited in their utility, because various capsule neurodegenerative diseases, with potential applications
parameters, such as size, mechanical strength, perm- in Parkinson’s and Alzheimer’s diseaxs. However, the
eability and wall thickness, are not independent of feasibility and chronic effects of long-term treatment
each otherqi. However, a new method, based on with these CNTF-producing capsules need to be
sodium alginate, cellulose sulphate, polymethylene- ascertained.
co-guanidine, CaCI, and NaCl, has the advantage of In addition, CNTF has recently been employed for
allowing independent modifications to the capsule the treatment of ALS’. Baby-hamster-kidney cells,
parameters and has recently been employed to encap- genetically engineered to secrete hCNTE were encap-
sulate rat Langerhans islets”. This formulation, with sulated in hollow-fibre membranes of poly-ether-
living cells transplanted into the peritoneal cavity of sulfone and implanted into the lumbar intrathecal space
mice, produced an effect in both chemically induced of six patients with an early stage of AL!?. The pro-
diabetic rats and in nonobese-diabetic (NOD) females; duction of hCNTF was detected in the cerebrospinal
these cells functioned for four to six months in the fluid of patients for up to 17 weeks’, and the devices
chemically induced diabetic rats and for LIP to six were removed 13 or 17 weeks following implantation”.
months in the NOD females+‘. This type of therapy The major side effects associated with systemic
offers the advantage of controlled secretion in response administration of hCNTF were not observed with this
to metabolic demands and signals, thus mimicking the form of delivery’. However, despite these encourag-
actions of functional cells, without the usual problems ing results, the disease continued to progress in these
associated with transplantation. Nonetheless, the rat patients and long-term studies involving a larger
islets failed, owing, not to rupture of the capsules or number of patients need to be conducted. Never-
an immune attack41, but possibly to the low viability theless, this mode of administration using genetically
of the islet cells themselves, resulting from the insults engineered cells holds promise for the treatment
sustained during their isolation from the pancreas. of neurodegenerative conditions. The use of non-
No treatment is presently available for Huntington’s biodegradable hollow fibres, which must be retrieved
disease (HD), which is characterized by cognitive and post-implantation, represents an alternative to micro-
motor deterioration and marked behavioural particles and microcapsules.
changeslo. CNTF has been shown in rodent models Cells from the human embryonic kidney cell line
of HD to prevent the loss of striatal neurons and 293, which produces rhCNTE were encapsulated in
to shield against behavioural deficienciesiO. Baby- biodegradable alginate-chitosan microspheres using an
hamster-tibroblast cells, genetically engineered to pro- extrusion method without the use of organic solvents3’
duce human CNTF (hCNTF), were encapsulated in and sustained in ~ifr~) drug delivery \vas achieved. The
asymmetrical hollow fibres of poly(acrylonitrile-co- use of long-lasting but biodegradable microspheres has
vinyl chloride) and implanted intrastriatally into cyno- the advantage over other invasive devices, such as can-
molgus monkeys; deterioration due to HD was imitat- nulae and hollow fibres, of not requiring retrieval after
ed using quinolic acid (QA) injection one week after implantation3’. These initial promising results need
implantation”‘. The animals were sacrificed three to be tested irz vivcj to determine the feasibility of
weeks after QA injection and the capsules retrieved these microspheres for the long-term treatment of
prior to death. In Nissl-stained sections of the stria- neurodegenerative conditions.
turn, a significant reduction of lesioned areas in the Recently, LM-10 tibroblast-like cells producing
caudate and putamen was observed in the hCNTF- rhCSF-1 have also been encapsulated in alginate-
treated monkeys compared with the controls’“. chitosan microspheres and then implanted at the site
Similarly, significant protection of glutamic acid of a unilateral cerebral cortex ischemic injury or in
decarboxylase-, choline acetyl transferase- and the peritoneal cavity in mice with no endogenous
NADPH-d-positive neurons was also provided by the CSF-13s. The microspheres with LR4-10 cells poten-
hCNTF-producing capsulesr(‘. In addition, atrophy of tiated microglial response to the injury, resulting in
neurons in layer V of the motor cortex ipsilateral to significant neuron rescues”. The survival time of the

TIBTECH OCTOBER 1997 NOL 15)

 417

reviews

LM-10 cells within the alginate-chitosan microspheres capsules in viva have still to be conducted in many
can be controlled by varying the physical properties of cases in order to determine the feasibility of sustained
the chitosan3*. These results show promise for the admin- drug release and possible long-term side-effects.
istration of cytokines or other neurotrophic factors to Recent progress in polymer science improving
the CNS in a sustained-release formulation and for the block copolymers and lipophilic, adhesive and bio-
use of cells producing such factors. erodable polymers will enable further development of
improved delivery systems useful for numerous
Future prospects medical applications.
Despite huge advances in the field of encapsulation Some of the problems associated with tissue-specific
of proteins and genetically engineered cells, many and site-specific delivery for long-term treatment of
problems remain to be solved. In the treatment of neurological disorders could be overcome by admin-
neurodegenerative conditions, some of the substances istration of multipotent neural progenitors or stem
that could provide rescue or treatment have been iden- cells. These cells could distribute a therapeutic gene
tified, but no suitable means of their delivery is in rou- product in a sustained and direct fashion throughout
tine clinical use. Some of these problems could be cir- the nervous system. Moreover, they may replace dys-
cumvented by the use of small cyclic peptides, but even functional neurons and glia in both a site-specific and
these molecules need to be delivered in a suitable man- global manner, thus offering alternative strategies for
ner’?. In comparison with the more established meth- treating neurodegenerative disorders45,4h,47.
ods of administering trophic factors, such as repeated
injections or via osmotic minipumps, neurotrophin Acknowledgments
delivery systems based on genetically engineered cells We wish to thank C. Allen for her contribution t:o
have several advantages. Firstly, neurotrophins stored the block-copolymer discussion and L. Zhang and
in a reservoir may be degraded over an extended A. Eisenberg for providing illustrations of block-
period of time, whereas cells can be engineered to copolymer micelles.
produce neuroactive agents continually. Secondly,
while only a finite amount of neuroactive agent can References
be stored and delivered in a reservoir, it is possible to
adjust the number of cells per sphere in order to
modulate the quantities secreted. Thirdly, most pumps 2 Gregorudis, G. (1995) 7’rendx Biotechvol. 13, 527-537
3 Las~c, D. D. (1996) Sa. Med. 3. 31-43
are only functional for up to two weeks and have to
4 Radler, J. O., K&over, I., Saldxtt, T. .uxd Safinya, C R. (1997)
be replaced if the treatment is required for longer Stre,tte 275. 81w311
periods of time; this results in greater subject morbid- 5 Allen, T. M. (1994) Trtwds Ph~nnaroi. Sn’. 15, 215-220
ity by, for example, increasing the risk of infection. 6 Puyal, C., Milhaud, P., B~envenoe, A. and Phibppot, J. R. (194’5)
However, cells can produce neuroactive agents for a Eur.). Bidrev. 228, 697-703
much longer period of time and are replaced less fre- 7 Maysiiger, D., Plccxdo, P. and Cuello, A. C. (1995) Rev. Aktrroxi 6,1%33
quently; moreover, recent developments in molecular 8 Aeblscher, P. and Tan, S. A. (1996) C Ibn F owrd. Symy. 196. 21 l-239
9 Aebischer, P. et ai. (lYY6) Xur. illed. 2, 696-699
biology suggest that we are now in a position to
10 Emench. D. F. et al. (1997) .Uturc 380, 395-399
improve the control of cell function and survival. 11 Donbrow. M., ed. (1 YY1) &ficrucapsrtles arzd N~nvparricles in .2lcdisinc
Due to the ability to control the release of agents and Nuwmy, CRC Press
microencapsulated by judicious choice of the materials 12 LI, Q., LIunn, T., Grandmaison, E. W. and Goosen, M. F. A. (19Y2)
of construction, this type of technology has the poten- J. Bioacr. Compaf. I’ofym. 7, 37+397
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ous injections, such as vaccination. Recently, oil-based Isvat. 98, 1117-1422
14 Chu, C. C. (1985) P+ncriz~!ron 26, 591-594
PLGA microcapsules fabricated by the solvent-
15 Miller, R. A., Brady, J. M. and Cutnght, D. E. (1977)) Biorncd.
evaporation method have been described that possess &later. Rer. 11, 711-719
kinetic properties that allowed the release of tetanus 16 Domb. A. J. (1994) in Polymeric Site-@@ Pharmacothcrqy
toxoid after three weeks or seven weeks-l”. The injec- (Domb, A. J., rd.), pp. l-26, John Wiley 8r Sons
tion of a cocktail of these microspheres could poten- 17 Cutnght, 1). E., Perez, B., Beasley. J. D., Larson, W. J. and
tially result in a pulsed release of the tetanus toxoid, Posey, W. R. (1974) Orai Sq. 37, 142-152
18 Sten&ichel, A. and Valentm, H. E. (1995) FElZlS,2licrobioi. Left. 128,
mimicking the action of the repeated injections
219-228
presently required to produce antibodies. In viva stud- 19 Miiller, H-M. and Seebach, D. (1993) A112ew. Chern., 1st. Ed. tzjj.
ies have still to be performed to assess whether the 32, 477-502
pulsed delivery from the microspheres will offer 20 Blssery, M-C.. Valenote, F. and Thws, C. (1984) III .bfkqdwres md
immunoprotection 44. This type of vaccination has the Drug Tkqq. Phamateutical, Itn,nu~olo~iial and ivled~tal ilspetts
potential to reduce costs and improve patient compli- (Davis, S. S., Ilium, L., McVle, J. G. and Tomlmson, E., eds),
pp 217-227, Eltrvirr Science
ance. Similar kinds ofbiodegradable microspheres may
21 Zhang, L., Yu, K. and Eisenberg, A. (1996) Science 272, 1777-1779
prove useful for drug delivery to the nervous system.
22 Zhang. L. and Eisenberg, A. (lY96)]. .+n. Chcm. Soc. 118,316s3181
Many techniques using microcapsules and micropar- 23 Zhang, L. and Eisenberg, A. (1995) Sderrce 268. 1728-1731
titles have produced sustained drug delivery in tlitro, 24 Kabanov, A. V. and Alakhov V. Y. in Anlphiphilic Block Cupolymers:
and a number of formulations have been tested in ho. Se!f-asrewbly atfd ilpplir&rt (Alexandris, P. and Lindman. B., edc),
Long-term trials of the microparticles and micro- Elsevier (m press)

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Emerging tandem-mass-
spectrometry techniques for the
rapid identification of proteins
Ashok R. Dongr6, Jimmy K. Eng and John R. Yates III

State-of-the-art techniques such as liquid-chromatography-electrospray-ionisation

tandem mass spectrometry have, in conjunction with database-searching computer
algorithms, revolutionised the analysis of biochemical species from complex
biological mixtures. With these techniques, it is now possible to perform h,igh-
throughput protein identification at picomolar to subpicomolar levels from protein
mixtures. This article provides an overview of the techniques and methodologies
available for the structural elucidation and identification of proteins and peptides
from complex biological samples.

The past ten years have seen an exponential growth in biological processes. These processes, such as antigen
the field of biological mass spectrometry. The incep- processing and presentation, covalent modifications
tion of such ‘soft’ ionisation techniques as fast-atom associated with signal transduction and single point
bombardmentlJ, electrospray3 and matrix-assisted mutations, can now be readily studied using tandem
laser desorption ionisation (~JIALDI)~*~ in the mid- mass spectrometry7-“. The worltdwide large-scale
1980s made it feasible to ionise and introduce into the DNA sequencing efforts that are currently being pur-
gas phase thermally labile compounds such as proteins, sued will further aid in the advancement of mass spec-
peptides, carbohydrates and oligonucleotides. These trometry to study the complex biological processeslO.
advances in ionisation methods, coupled with various The complete genomic analyses of organisms such as
mass analysers, have generated new, technologically Haemophilus influenzae, Saccharomyces cevevisiae and
sophisticated, mass spectrometry instrumentation that is Escherichia coli, and the impending complete sequenc-
currently being used to characterise the primary struc- ing of the human genome, will create rich genome-
tures of peptides and proteins involved in complex sequence databases 11 that will enhance our ability to
study both physiological and biochemical processes.
A. R. Don@ (adoHgre@u.washington.edu}, J. K. Eng (eqj@ The primary goal of a biological study is to identify
u.washington.edu) and J. R. Yates III liyates@u.washiH~~tor2.edu) the functional species involved in a process, and
are af the Deparrmetit of A4okrulav Biotechnology, University of
involves placing a biological context on genes and gene
Washington, Seattle, WA 98195, USA. A. R. Dar@ is also al the
Department ofImmunology and the Howard Hughes Medical Institute products. Tandem mass spectrometry is ideally suited
al the University of Washiqgton, Seattle, WA 98195, USA. to provide a powerful link between genomics

TIBTECH OCTOBER 1997 NOL 15) Copynght Q 15197, tlsevler Sctence Ltd. All rights reserved. 0167 - 7799/97/$17.00. HI: SO167-7799(97)0111@l