- Email: [email protected]
Drug delivery to the nervous system
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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 neurodegenerative
of the mechanisms
disorders
involved in the development
and the actions of neuroactive
of
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 from the lack of simple hormones or peptides, the administration of these compounds in a controlled fashion could provide therapy. Conditions such as diabetic neuropathy, amyotrophic lateral sclerosis (ALS) and Huntington’s disease, and possibly also Alzheimer’s and Parkinson’s diseases, could benefit from this type of approach. Nevertheless, although some of the candidate compounds for treatment have been identified, an adequate means of long-term delivery to the nervous system is still lacking. Only a limited number of small, stable molecules acting on neurons or nonneuronal tissue are available for long-term treatment that can be administered orally. However, peptide drugs and small peptides are easily degraded by proteolytic enzymes and hence cannot reach their site of action when administered in this way. The strategy for the administration of these readily hydrolysable molecules for the treatment of neurodegenerative conditions of the central and peripheral nervous system (CNS and PNS, respectively) can assume two directions - either the defective or absent gene is replaced, with the expectation that the missing compound will then be produced, or the missing compound is provided directly. This article focuses on the latter approach, which can be achieved by direct delivery of the molecule or through the use of living cells that produce the missing substance. D. Maysinger [[email protected]) and A. Morinville are at the Department $Pharmacology and 7’herapeutics, McGill University, 3655 Drummond Street, Room 13 14, Montreal, Quebec, Canada H3G lY6. TIBTECH OCTOBER 1997
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Elsevier
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Drug-delivery systems ‘Microcapsules’ contain an agent in their centre surrounded by a polymer or a macromolecular carrier. A drug (or other active agent, or a living cell) is in the core of the microcapsule and the surrounding polymer layer can vary in thickness and permeability, and their diameter usually ranges between 1 and 2000 micrometresi. Microcapsules are also known as microspherules, spansules, and coate’d granules, pellets or seeds’, and can have several separate cores surrounded by a polymer or a mixture of polymers. ‘Microparticles’ are matrices containing drugs (or other agents) more or less homogeneously distributed (suspended) throughout the entire matrix with no distinct core or envelope. Their dimensions are in the micrometre to nanometre range. ‘Microspheres’ (nanospheres) represent examples of microparticles (nanoparticles). Examples of microparticles and microcapsules and other delivery devices containing neuroactive agents or living cells for administration to tlhe CNS and PNS are provided in Table 1. Targeted drug delivery and the related problems Two main categories of targeting exist - passive and active. Passive targeting occurs because of the body’s natural responses to foreign material; macrophages from the liver and the spleen play the pivotal role in these actions. Small particles are not as subject to reticuloendothelial-system (RES) uptake as larger ones, and polymeric materials can also influence this process - for example, hydrophilic, neutral surfaces are favoured over more lipophilic larger particles. In Ltd. All rights reserved.
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reviews contrast, active targeting may involve the attachment of a receptor device or vector molecule to the drugdelivery system. If the target site is the liver or spleen, the drug-delivery vehicle should be designed to activate the complement system in order to promote RES uptake. If the target is another organ, the vehicle must avoid RES uptake and should include a vector molecule that will direct it to this organ. However, this task remains difficult and only limited success has been achieved so far.
Liposomes
Drug-delivery devices Microcapsules Microsphere-matrix type Liposomes Micelles Conjugates
Gelforms Collagengels Osmoticmini-pumps Adhesivepatches
Living-cell-delivery devices
Since their discovery by A. D. Bangham in the 196Os’, liposomes have received much attention as drug carriers. Conventional liposomes resemble plasma membranes, consisting of phospholipid molecules with a polar head with two hydrophobic tails, forming a bilayerj. In contrast, micellar subunits possess one such tail and form a single layer3. Liposomes are biocompatible, nontoxic and biodegradable2J,“, and offer the possibility of carrying hydrophobic, hydrophilic or amphiphilic molecules3. Despite these numerous attributes, liposomes have not gained widespread use as drug-delivery systems, due to their instability in viv$J,“. Classical liposome preparations, sterically stabilized liposomes and immunoliposomes are all susceptible to clearance by the immune system’J,j. Most work on liposomal applications has centred on the delivery of anticancer agents and has culminated in the liposomal formulation of doxorubicin3.5. Recent advances in liposomal formulations include cationic liposomes used to entrap various polynucleotide@. In addition, cationic liposomes have been complexed with DNA; these are synthetically based nonviral carriers of DNA vectors for gene therapy”, but the in vim lifetime of these promising formulations remains to be determined. At present, the working in vivo range of liposomes is restricted to the nanometre scale2 as long-term circulation depends on size: the larger the liposome, the faster the clearance. Because the action of neuroactive agents (for example, neurotrophic factors) is mostly achieved by their coupling to a receptor on the plasma membrane, causing an intracellular cascade of events, the formulation of these molecules for delivery must allow sustained extracellular release. Sterically stabilized liposomes, which are taken up by endocytosiss, are therefore unsuitable for this purpose. Finally, owing to the often potent nature of neuroactive agents, sustained drug release, entailing extended in vim stability of the delivery system, remains difficult with liposomes. In conclusion, liposomes are presently unsuitable for the long-term administration of drugs to the CNS and PNS unless adequately stabilized. Microcapsules and microparticles A delivery device for long-term treatment nervous system needs to possess a lifetime that that of the entity it is carrying, which may example, a peptide molecule or a living cell. the difficulties in penetrating the blood-brain
Table 1. Delivery of drugs and cells to the central and peripheral nervous systems
of the exceeds be, for Due to barrier,
Encapsulatedcells Injectionof dispersedcells
Hollowfibres Collagengels
research into the long-term administration of drugs to the CNS has classically been directed at permanently installed devices. In order to avoid inconvenient inse:rtions and the possible removal of large implants or permanently installed stainless-steel cannulae, several types of injectable drug formulations, including microcap sules and microparticles, have been applied in neuroscientific research, showing some promise for clinical use. On the other hand, systemic or local drug delivery is needed for administration to the PNS, for example in diabetic neuropathy. Regardless of the ultimate goal, one of the major obstacles remains the choice of construction materials; the biocompatibility and controlled rate of drug release of the material are impo:rtant variables in the selection process. Table 2 lists some examples of materials used for the encapsulation of neuroactive agents. Polymers that can be used for the preparation of implants or microspheres may be either biodegradable or nonbiodegradable, with both types having eno-rmous potential for drug delivery in the CNS and for use as vehicles for transplanting viable cells7.8. Polysulfones’, poly(acrylonitrile-co-vinyl chloride)*O, ethylene-vinyl acetate (EVA) and hydroxyethylmethacrylate-methyl-methacrylate copolymer (HEMA-MMA)7,8 are frequently used nonbiodegratlable materials. These water-insoluble systems yield capsules that are mechanically and chemically stable and are convenient for cell implantation’. The pol;rmer acts as a semipermeable membrane, allowing the diffusion of the neuroactive agent. However, encapsulation of living cells using these types of materials pnsents numerous difficulties because of toxic effects of solvents on cells, either during the formation of thermoplastic-based capsules or because of residual solvent. Biodegradable polymers can be either synthetic or natural and may be degraded in vivo both enzymatitally and nonenzymatically. Their byproducts should be biocompatible, nontoxic and readily excreted. Only a few immunologically tolerable biodegradable polymers are available. Human serum albumin and bovine serum albumin have been extensively used for target-’ ing anticancer drugs, insulin, and monosialoganglioside TIBTECH OCTOBER 1997 WOL 151
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reviews Table 2. Examples of materials used for encapsulation7.9JOJ3 Name
Source
Biodegradability
Comments
Humanserumalbumin, bovineserumalbumin Collagen,haemoglobin, gelatine,chitosan,alginate andpoly-L-lysine PolyOactide-co-glycolide)
Natural
Biodegradable
Natural
Biodegradable
Nontoxic, immunologically tolerable Nontoxic, immunologically tolerable,doubtfulpurity
Synthetic
Biodegradable
Polyhydroxyalkanoate
Naturalor synthetic Biodegradable
Ethyl-vinylacetate, Synthetic hydroxyethyl-methacrylatemethyl-methacrylate copolymer Polysulfones, Synthetic polyfacrylonitrile-co-vinyl chloride) Fumaricandsebacicacids Synthetic
Nonbiodegradable
Versatile,controlleddrug release, erodable Thermoplastic,widevariety of repeatingsubunits,controlled drugrelease Thermoplastic
Nonbiodegradable
Retrievalafter implantation
Biodegradable
Adhesivemolecules,increased in viva lifetime,usefulfor drug deliveryto gastrointestinaltract
(GMl). Other natural biopolymers, such as collagen, haemoglobin, gelatine, chitosan, alginate and poly-Llysine, have been employed as carriers for parenteral administration’t.la.13, but their cost and lack of purity have hindered their use. Therefore, several types of synthetic biodegradable polymers have recently been developed and used for microencapsulation, because their processing conditions, availability and cost can be controlled more efficiently’. Among the biodegradable polyesters, which hold considerable promise for the controlled delivery of proteins, are those based on poly(lactic acid) and poly(glycolic acid). Poly(lactide-co-glycolide) (PLGA), commonly used in sutures, provides great flexibility in terms of composition, thus allowing tailored drug release. The biodegradation and tissue reactions of PLGA have been extensively investigatedr”,ls. PLGA is a linear polyester prepared by ring-opening polymerization and subsequently purified to yield a with the chemical structure polymeric powder [-H2C-CO-O-],, [-CH(CH3)-CO-O-],,, and it hydrolyses by an acid- or base-catalysed reaction to form lactic and glycolic acids. The degradation properties of PLGA depend on the molar ratios of the two monomers in the polymer chain, the molecular mass, crystallinity, size and shape of the device, and on the implantation site 16. For example, the time taken for PLGA copolymer to biodegrade can be varied from about 30 days to almost a year1ss17 by adjusting the lactic acid:glycolic acid ratio. Polyhydroxyalkanoates (PHA) represent another class of biodegradable materials amenable for microencapsulation and are potentially superior to PLGA. These biodegradable plastics are synthesized by a number of bacterial species’s,‘“. The majority of PHAs contain from three to six carbon atoms in the polyTIBTECH OCTOBER 1997
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ester backbone, with numerous va:riations in the Rpendant chain’*. Most poly-R-(-)-Shydroxyalkanoic acids are composed of a three-carbon-atom backbone and up to eleven carbon atoms in the pendant chainIs. The structure for these poly-R-(-)-3-hydroxyalkanoic acidslsJ” is [-0-CHR-CH,-CO-O-],, where R represents the pendant chain. This variation in repeating units confers a greater flexibility for tailoring the agent-release properties compared to PLGAs. Because they are part of the natural biosynthesisbiodegradation cycle, PHAs have few or no side effects and, under aerobic conditions, they degrade to water and carbon dioxide’“. The most widely used of these thermoplastic esters is poly(3-hydroxybutyrate), commonly abbreviated to P(3-HB) or PHB. PHB is often employed in conjunction with hydroxyvalerate to form copolymers such as poly(3hydroxybutyrate)(3-hydroxyvalerate), P(3HB-3HV). PHAs have been employed to microencapsulate, among other things, anticancer drugs such as l-(2 chloroethyl)-3cyclohexylnitrosourea (CCNU)20 and 2’,3’-diacyl-5fluoro-2’-deoxyuridine. Block-copolymer micelles in drug delivery Traditionally, micellar drug-delivery systems have been formed from small-molecule surfactants and amphiphilic block-copolymer molecules. The incorporation of the drug into small-molecule surfactant micelles is quite low, representing one of their major limitations as successful delivery systems. In contrast, block-copolymer micelles can incorporate agents to a greater degree. Block-copolymer micelles are formed from individual block-copolymer chains containing a hydrophobic block and a hydrophihc block. A variety of shapes and sizes (between 10 and 100 nm) can be produced using technologies developed by Eisenberg
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reviews
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 favourable for avoiding capture by the RES. The amphiphilic character of the block copolymers enables selfassembly of nanometer-scale aggregates with a hydrophobic core and a hydrophilic outer shell in aqueous media. The hydrophobic core accommodates nonpolar drugs and the properties of the hydrophilic shell can be adjusted to both maximize biocompatibility and avoid RES uptake. Three main groups ofblock-copolymer delivery systems exist - polymeric drugs, micellar microcontainers and block-ionomer complexes21. Polymeric drugs are block-copolymer-drug conjugates in which the drug is covalently bound to the polymer carrier. Common synthetic polymers and copolymers employed as carriers include poly(divinyl-ether-comaleic anhydride), poly(styrene-co-maleic anhydride) and homopolymers such as poly(L-lysine), poly(ethylene glycol) (PEG) and poly(L-aspartic acid). PEGpolyaspartic-acid block copolymers were used to bind adriamycine, and this system showed several advantages over adriamycine in non-micellar formulation. The major advantages were reduced binding to albumin, greater stability in vitro, lower systemic toxicity and apparent higher effectiveness as an anticancer
agent. However, the decreased cytotoxicity was caused by the slower release .of the covalently bound adriamycine from the polymeric Carrie?. Recently, the focus has been on micellar systems including noncovalently incorporated drugs, for example micellar microcontainers. These types of micelles are made of diblock and triblock copolymers, and in aqueous solutions they form a water-soluble shell (corona) with a micellar core formed &om the hydrophobic loops of polymer chains. The dibl’ock and triblock copolymer micelles are both therrnodynamically and kinetically stable in aqueous media and are promising drug-delivery vehicles. Block-ionomer complexes are formed from amphiphilic block copolymers with a charged hydrophobic core-forming block. The compound to be incorporated into these micelles must have the opposite charge from the core-forming block. When the block copolymer is mixed with a drug, a complex is formed because of electrostatic interactions between the core-forming block and the drug. This kind of micellar block-ionomer complex has recently been shown to be useful for DNA complexing, allowing a novel approach to DNA delivery consisting of the formation of DNA-block-ionomer complexes. Kabanov TIBTECH OCTOBER 1997
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reviews et al.*” demonstrated enhanced stability of 19-mer oligonucleotide incorporated into micelles of poly(ethylene oxide)-b-polyspermine in serum. Such types of ionomer-micelle complexes are potentially useful as delivery systems in gene therapyad. The therapeutic potential of many drugs, particularly those that are highly liposoluble, could be greatly enhanced by an adequate selection of block-copolymer micelles. The progress in polymer chemistry, in particular block copolymers and rationally designed small peptides that mimic the effects of large neurotrophic proteins, may offer new ways to deliver drugs to the PNS and CNS. Biologically adhesive molecules An exciting new range of materials for microspheres are the so-called biologically adhesive molecules. These molecules possess advantages over the other drug-carrier systems, in that they exhibit increased residence time in vioo and, hence, the effects of the drug they carry could be sustained for longer intervals. Traditional approaches to the development of biologically adhesive molecules have centred on the use of hydrophilic polymers and hydrogels containing carboxyl groupsz5. Recently, a rapidly degrading hydrophobic polymer consisting of polyanhydride copolymers of fumaric and sebacic acids (FA:SA) exhibited delayed passage through the gastrointestinal tract when compared with other polymers due to an increase in biological adhesiona5. Poly(FA:SA) 20:80 nanospheres 4ct120 nm in size, fabricated using phase-inversion nanoencapsulation, were detected in the cytoplasm of cells and also inside the Golgi apparatus and secretory vesiclesas. Within the poly(FA:SA) nanospheres, dicumarol, a low molecular weight compound with low solubility and poor bioavailability, displayed increased bioavailability in animals compared with other modes of oral administration’j. Similarly, orally delivered insulin, encapsulated in a 50:50 blend of poly(fumaric anhydride) and PLGA improved the regulation of glucose in rats in comparison with rats fed an insulin in saline solution or a saline solution alone25. These results should, however, be interpreted with caution, as the animals were not diabetic and the usual parenteral administration of insulin was not assessed in comparison with the oral nanosphere formulation. Systemic circulation of these bioadhesive molecules, required for the treatment of many conditions, was not demonstrated. Despite this, these preliminary studies offer exciting possibilities for the oral delivery of a variety of compounds.
Delivery of neuroactive agents ‘Neuroactive agents’ is a broad term, and includes several categories of peptide, protein and small molecules that act on the nervous system. Neurotrophic agents are substances capable of producing reparation, regeneration or protection of damaged cells in the nervous system. ‘Neurotrophic factors’ are endogenously produced peptides that participate in the establishment and maintenance of the neural phenotype development and adulthood. during embryonic TIBTECH OCTOBER 1997 (VOL 15)
‘Neurotrophins’ comprise a restricted group of neurotrophic factors that are structurally related to nerve growth factor (NGF). Strategiesfor delivery Various approaches have been developed to provide continuous delivery of neuroactive agents, and, although these have overcome some of the problems of delivering these agents, numerous additional problems remain. Even though drug delivery to the PNS appears simpler than delivery to the CNS, problems such as the site of administration, linearity of release, biocompatibility of the polymeric material and loading capacity have still to be solved. F’ermeabilizers of the blood-brain barrier, osmotic pumps, slow-release polymeric devices and particles, and igenetically engineered cells represent some of the tested means of neuroactive-agent delivery. In addit:lon, transfecting neural cells in vivu with viral vectors has been extensively investigated and certainly deserves attention (for reviews, see Refs 26-28). Table 3 summarizes some of the advantages and disadvantages of the main categories of nonviral delivery systems. The encapsulation of growth factors and of cells engineered to secrete them is in its infancy; so far, only a limited number have been tested in in uivo models (rodents and primates)y~ay-31. Several recently described examples of neurotrophin administration using microcapsules and microparticles will be summarized here, with an emphasis on those factors claimed to be effective in rescuing or preventing neurodegenerative conditions. Essentially, two main approaches to the delivery of neuroactive agents in encapsulated formulations exist - biodegradable polymeric microparticles and microcapsules, and nonbiodegradable delivery devices such as hollow fibres that accommodate cells synthesizing trophic factors (see Table 1). Delivery of neuroactive agents using microcapsules and microparticles Among the most studied neurotrophins are NGF, brain-derived neurotrophic factor (BDNF), neurotrophins 3 and 4 (NT-3, NT-4) and glial-derived neurotrophic factor (GDNF). The physiological roles of these and related neurotrophic factors in nervoussystem development and degeneration8v32.33, the signaling mechanisms related to them31 and the problems of their delivery to the nervous system7,* have been extensively reviewed. The administration of growth factors such as NGE insulin-like growth factor (IGF) or human growth hormone (hGH) typically poses many problems, owing to limited oral bioavailability and short in uivo lifetimes3s. A number of human trials involving ciliary neurotrophic factor (CNTF) have yielded disappointing results36, often because of serious side effects (toxicity)37; improvements in drug delivery could lower the therapeutic dose and hence reduce these side-effects36. Sustained-release formulations involving microspheres composed of biocompatible and biodegradable polymers could overcome these
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reviews Table 3. Properties of common nonviral delivery systems for the peripheral and central nervous systems* Nonviral delivery Site of action (central Advantages system or peripheral nervous system)
Disadvantages
Blood-brainbarrier Central permeabilizers
Highsystemicdoses,unlocalized braindelivery
Osmoticpumps
-
Systemicuptake
Centraland peripheral Cerebrospinal-fluid delivery, long-termdelivery
Failurecould leadto potentiallytoxic 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 compounds. Recombinant human CNTF (rhCNTF), which has possible applications in the treatment of ALS, was encapsulated in PLGA-and-chitosan microspheres and also in dry alginate with chitosan using the phase-evaporation method37. Sustained release was achieved in both cases, with a higher rate of release for the alginate-chitosan microspheres (2-l 2 days), whereas with PLGA-chitosan microspheres, drug release was observed up to 24 days37, but this formulation has still to be tested if2 viuo. Colony stimulating factor 1 (CSF-1) has been implicated in the differentiation, proliferation and survival of microglia, but has typically posed delivery problems caused by enzymatic degradation38. Recombinant human CSF-1 (rhCSF-1) was encapsulated in PLGA-chitosan microspheres by the solvent-evaporation method3*. These microspheres potentiated the microglial response to injury; the effect was slightly less when the microspheres were implanted in the peritoneum rather than at the site of lesion3R. Treatment of diabetic neuropathy could benefit from the encapsulation of neuroactive agents. Reduction of IGF activity has been shown in both human and nonhuman models of diabetesj9. The expression of IGF-I is controlled by and mimics that of hGH, decreasing after the third decade in nondiabetic individuals3”13”. Recently, recombinant human growth hormone (rhGH) has been encapsulated in PLGA microspheres using a cryogenic process35. In monkeys, injection of these microspheres resulted in an increase of both rhGH and IGF-I for a period of one month3”, even though daily injections of rhGH failed to elicit the same response35. Therefore, growth factors within microspheres represent an exciting combination for the potential treatment of neurodegenerative conditions of the PNS.
Living cells for drug delivery Many neurodegenerative conditions could pot’entially be treated by the administration of neuroactive agents in a controlled manner through the use of living cells, either wild-type or genetically engineered, that secrete these molecules. However, immortalized cells grafted into the CNS often give rise to tum’our formation in the host tissue’; the use of primary cells is a possible strategy to overcome this problem. Nevertheless, cell transplantation has typically posed problems due to the low availability of human cells and immunorejection 41, leading to lifelong treatments with immunosuppressants. Immunoisolation offers the advantages of reducing immunorejection ;and introducing the possibility of xenograftsJ1. The immunoisolating device must allow the passage of nutrients and oxygen as well as the active compound of interest, and must be biocompatible with both the host and the transplanted cell. Microencapsulated cells may thus become efficient biological pumps, overcoming some of the difficulties associated with the implantation of dispersed cells or repeated injections ofdrugs (Table 3). PC12 pheochromocytoma cells .and fibroblasts are commonly used in these delivery systems. Encapsulated cells have improved survival .and sometimes even produce more neurotrophin t:han when grown as monolayer culturesZ9.30,37. Anchoragedependent cells such as fibroblasts need a support to provide long-term survival, and matrigel, collagen and polyelectrolytes (alginate, chitosan) are commonly used inside the polymer capsules. The use of hollow fibres of nonbiodegradable polymers has the advantage of allowing the device to’ be retracted when it is not needed or when the cells cease to produce the drug, although surgery would be necessary. In some cases, the administration of neurotrophins or neurotrophin mimetics”* in biodegradable TIBTECH OCTOBER 1997
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reviews microspheres is advantageous, because the polymer will gradually erode, yielding biocompatible degradation products that can easily be eliminated by the body without the need for surgical removal. Technological approaches to and the current status of nonbiodegradable hollow-fibre encapsulation have been extensively discussed in recent reviewssJ3. Delivery using encapsulated living cells A variety of devices have been developed for the delivery of living cells, including microcapsules, macrocapsules, h o 11ow fibres, tubular membranes, intravascular devices and flat sandwich pouchesAl. The usual construction materials, such as HEMA-MMA or poly-L-lysine with hardened polyanionic gel, are limited in their utility, because various capsule parameters, such as size, mechanical strength, permeability and wall thickness, are not independent of each otherqi. However, a new method, based on sodium alginate, cellulose sulphate, polymethyleneco-guanidine, CaCI, and NaCl, has the advantage of allowing independent modifications to the capsule parameters and has recently been employed to encapsulate rat Langerhans islets”. This formulation, with living cells transplanted into the peritoneal cavity of mice, produced an effect in both chemically induced diabetic rats and in nonobese-diabetic (NOD) females; these cells functioned for four to six months in the chemically induced diabetic rats and for LIP to six months in the NOD females+‘. This type of therapy offers the advantage of controlled secretion in response to metabolic demands and signals, thus mimicking the actions of functional cells, without the usual problems associated with transplantation. Nonetheless, the rat islets failed, owing, not to rupture of the capsules or an immune attack41, but possibly to the low viability of the islet cells themselves, resulting from the insults sustained during their isolation from the pancreas. No treatment is presently available for Huntington’s disease (HD), which is characterized by cognitive and marked behavioural deterioration and motor changeslo. CNTF has been shown in rodent models of HD to prevent the loss of striatal neurons and to shield against behavioural deficienciesiO. Babyhamster-tibroblast cells, genetically engineered to produce human CNTF (hCNTF), were encapsulated in asymmetrical hollow fibres of poly(acrylonitrile-covinyl chloride) and implanted intrastriatally into cynomolgus monkeys; deterioration due to HD was imitated using quinolic acid (QA) injection one week after implantation”‘. The animals were sacrificed three weeks after QA injection and the capsules retrieved prior to death. In Nissl-stained sections of the striaturn, a significant reduction of lesioned areas in the caudate and putamen was observed in the hCNTFtreated monkeys compared with the controls’“. Similarly, significant protection of glutamic acid decarboxylase-, choline acetyl transferaseand NADPH-d-positive neurons was also provided by the hCNTF-producing capsulesr(‘. In addition, atrophy of neurons in layer V of the motor cortex ipsilateral to TIBTECH OCTOBER 1997 NOL 15)
the lesion was reduced in animals with the hCNTF cells’“. The retrieved implants exhibited a marked decrease in the rate ofsecretion of CNTF with respect to the levels measured prior to implantation (12.7 versus 45.1 ng dayi capsule-‘)I”. The reason for the decreased production was not determmed and the state of the retrieved capsules was not asses:sed.Death of the encapsulated cells would lead to decreased production; this could be caused by insufficient nutrient availabilirq an immune response to the cells or a reduction in cell viability due to the encapsulation conditions. Nevertheless, the neuroprotective efIect on the cortical neurons innervating the striatum generated by these encapsulated hCNTF-producing cells provides encouraging results for the treatment of HD and other neurodegenerative diseases, with potential applications in Parkinson’s and Alzheimer’s diseaxs. However, the feasibility and chronic effects of long-term treatment with these CNTF-producing capsules need to be ascertained. In addition, CNTF has recently been employed for the treatment of ALS’. Baby-hamster-kidney cells, genetically engineered to secrete hCNTE were encapsulated in hollow-fibre membranes of poly-ethersulfone and implanted into the lumbar intrathecal space of six patients with an early stage of AL!?. The production of hCNTF was detected in the cerebrospinal fluid of patients for up to 17 weeks’, and the devices were removed 13 or 17 weeks following implantation”. The major side effects associated with systemic administration of hCNTF were not observed with this form of delivery’. However, despite these encouraging results, the disease continued to progress in these patients and long-term studies involving a larger number of patients need to be conducted. Nevertheless, this mode of administration using genetically engineered cells holds promise for the treatment of neurodegenerative conditions. The use of nonbiodegradable hollow fibres, which must be retrieved post-implantation, represents an alternative to microparticles and microcapsules. Cells from the human embryonic kidney cell line 293, which produces rhCNTE were encapsulated in biodegradable alginate-chitosan microspheres using an extrusion method without the use of organic solvents3’ and sustained in ~ifr~) drug delivery \vas achieved. The use of long-lasting but biodegradable microspheres has the advantage over other invasive devices, such as cannulae and hollow fibres, of not requiring retrieval after implantation3’. These initial promising results need to be tested irz vivcj to determine the feasibility of these microspheres for the long-term treatment of neurodegenerative conditions. Recently, LM-10 tibroblast-like cells producing rhCSF-1 have also been encapsulated in alginatechitosan microspheres and then implanted at the site of a unilateral cerebral cortex ischemic injury or in the peritoneal cavity in mice with no endogenous CSF-13s. The microspheres with LR4-10 cells potentiated microglial response to the injury, resulting in significant neuron rescues”. The survival time of the
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reviews LM-10 cells within the alginate-chitosan microspheres can be controlled by varying the physical properties of the chitosan3*. These results show promise for the administration of cytokines or other neurotrophic factors to the CNS in a sustained-release formulation and for the use of cells producing such factors. Future prospects Despite huge advances in the field of encapsulation of proteins and genetically engineered cells, many problems remain to be solved. In the treatment of neurodegenerative conditions, some of the substances that could provide rescue or treatment have been identified, but no suitable means of their delivery is in routine clinical use. Some of these problems could be circumvented by the use of small cyclic peptides, but even these molecules need to be delivered in a suitable manner’?. In comparison with the more established methods of administering trophic factors, such as repeated injections or via osmotic minipumps, neurotrophin delivery systems based on genetically engineered cells have several advantages. Firstly, neurotrophins stored in a reservoir may be degraded over an extended period of time, whereas cells can be engineered to produce neuroactive agents continually. Secondly, while only a finite amount of neuroactive agent can 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 are only functional for up to two weeks and have to be replaced if the treatment is required for longer periods of time; this results in greater subject morbidity by, for example, increasing the risk of infection. However, cells can produce neuroactive agents for a much longer period of time and are replaced less frequently; moreover, recent developments in molecular biology suggest that we are now in a position to improve the control of cell function and survival. Due to the ability to control the release of agents microencapsulated by judicious choice of the materials of construction, this type of technology has the potential for use in a number of areas that require numerous injections, such as vaccination. Recently, oil-based PLGA microcapsules fabricated by the solventevaporation method have been described that possess kinetic properties that allowed the release of tetanus toxoid after three weeks or seven weeks-l”. The injection of a cocktail of these microspheres could potentially result in a pulsed release of the tetanus toxoid, mimicking the action of the repeated injections presently required to produce antibodies. In viva studies have still to be performed to assess whether the pulsed delivery from the microspheres will offer immunoprotection 44. This type of vaccination has the potential to reduce costs and improve patient compliance. Similar kinds ofbiodegradable microspheres may prove useful for drug delivery to the nervous system. Many techniques using microcapsules and micropartitles have produced sustained drug delivery in tlitro, and a number of formulations have been tested in ho. Long-term trials of the microparticles and micro-
capsules in viva have still to be conducted in many cases in order to determine the feasibility of sustained drug release and possible long-term side-effects. Recent progress in polymer science improving block copolymers and lipophilic, adhesive and bioerodable polymers will enable further development of improved delivery systems useful for numerous medical applications. Some of the problems associated with tissue-specific and site-specific delivery for long-term treatment of neurological disorders could be overcome by administration of multipotent neural progenitors or stem cells. These cells could distribute a therapeutic gene product in a sustained and direct fashion throughout the nervous system. Moreover, they may replace dysfunctional neurons and glia in both a site-specific and global manner, thus offering alternative strategies for treating neurodegenerative disorders45,4h,47. Acknowledgments We wish to thank C. Allen for her contribution t:o the block-copolymer discussion and L. Zhang and A. Eisenberg for providing illustrations of blockcopolymer micelles. References
2 Gregorudis, G. (1995) 7’rendx Biotechvol. 13, 527-537 3 Las~c, D. D. (1996) Sa. Med. 3. 31-43 4 Radler, J. O., K&over, I., Saldxtt, T. .uxd Safinya, C R. (1997) Stre,tte 275. 81w311 5 Allen, T. M. (1994) Trtwds Ph~nnaroi. Sn’. 15, 215-220 6 Puyal, C., Milhaud, P., B~envenoe, A. and Phibppot, J. R. (194’5) Eur.).
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Maysiiger, D., Plccxdo, P. and Cuello, A. C. (1995) Rev. Aktrroxi 6,1%33 Aeblscher, P. and Tan, S. A. (1996) C Ibn F owrd. Symy. 196. 21 l-239 Aebischer, P. et ai. (lYY6) Xur. illed. 2, 696-699 Emench. D. F. et al. (1997) .Uturc 380, 395-399 Donbrow. M., ed. (1 YY1) &ficrucapsrtles arzd N~nvparricles in .2lcdisinc and Nuwmy, CRC Press 12 LI, Q., LIunn, T., Grandmaison, E. W. and Goosen, M. F. A. (19Y2) J. Bioacr. Compaf. I’ofym. 7, 37+397 13 Sun, Y., Ma. X., Zhou, D.. Vacrk. L and Sun, A. M. (lYY6)j. Cfin. Isvat. 98, 1117-1422 14 Chu, C. C. (1985) P+ncriz~!ron 26, 591-594 15 Miller, R. A., Brady, J. M. and Cutnght, D. E. (1977)) Biorncd. &later. Rer. 11, 711-719 16 Domb. A. J. (1994) in Polymeric Site-@@ Pharmacothcrqy (Domb, A. J., rd.), pp. l-26, John Wiley 8r Sons 17 Cutnght, 1). E., Perez, B., Beasley. J. D., Larson, W. J. and Posey, W. R. (1974) Orai Sq. 37, 142-152 18 Sten&ichel, A. and Valentm, H. E. (1995) FElZlS,2licrobioi. Left. 128, 219-228 19 Miiller, H-M. and Seebach, D. (1993) A112ew. Chern., 1st. Ed. tzjj. 32, 477-502 20 Blssery, M-C.. Valenote, F. and Thws, C. (1984) III .bfkqdwres md Itn,nu~olo~iial and ivled~tal ilspetts Drug Tkqq. Phamateutical, (Davis, S. S., Ilium, L., McVle, J. G. and Tomlmson, E., eds), pp 217-227, Eltrvirr Science 21 Zhang, L., Yu, K. and Eisenberg, A. (1996) Science 272, 1777-1779 22 Zhang. L. and Eisenberg, A. (lY96)]. .+n. Chcm. Soc. 118,316s3181 23 Zhang, L. and Eisenberg, A. (1995) Sderrce 268. 1728-1731 24 Kabanov, A. V. and Alakhov V. Y. in Anlphiphilic Block Cupolymers: Se!f-asrewbly atfd ilpplir&rt (Alexandris, P. and Lindman. B., edc), Elsevier (m press) TIBTECH OCTOBER 1997 NOL 15)
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reviews 25 Mathlolultz, E. et al. (1997) h’ature 386, 41@414 26 Zum, A. D., Tseng,J. and Aebischer, P. (1996) Eur. ~Veuml. 36,405408 27 Langer, R. (1996) iVat. .&f. 2, 742-743 28 Snyder, E. Y. and Fisher, L. J, (1996) Cur. Opin. Pediatr. 8, 558568 29 Aebischer, P.. Goddard, M., Signore, A. and Timpson, R. (19Y4) Exp. h’eurol. 126, 151-158 30 Llndner, M. D. et al. (1995) Exp. Meurol. 132, 62-76 31 Llberim, P., Ploro, E. P., MaysInger, D., Erwin, F. R. and Cuello, A. C. (1993) IVeuroxience 53, 625437 32 Lewin, G. R. and Barde. Y-A. (1996) A wu~. Rev. Neu~osci. 19,2X9-31 7 33 SegaI,R. A. andGreenberg, M. E. (1996)Annu. Rev. Neurosci. 19,463-4X1 34 Ip, N. Y. andYancopouIor, G. D. (1996) A nnu. Rev. Neurosti. lY,491-515 35 Johnson 0. L. et al. (1996) h’at. Med. 2, 795-799 36 Barinaga, M. (1994) S&m 264, 772-774 37 Maysinger, D., Krieglstein, K., Fihpovlc-Grcic, J., Sendtner, M.,
Unsicker,
K. and Richardson,
P. (1996) Exp. Xeuml. 138, 177-188 S. (1996) Exp.
38 MaysInger, D., Berezovskaya, 0. and Fedoroff, Xwol. 141, 47-56 39 Ishli, D. N. (1995) Brairl Res. Rev. 20, 4747
40 Le Roith, I). (1997) AVeujEngl.J Med. 336, 633-640 41 Wang, T. ct al. (1997) Nat. Biulerlmol. 15, 3!i8-362 42 LeSauteur, L., Wei, L., Gibbs, B. F. and Sal-agovi, U. (1995)J. Chem.
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Langer. 45 Snyder, 46 Snyder,
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6564-6569
P. J. (1996) Trozds Btotedzhnol. 14, 163-167 A., Gupta, R. K., Alonson, M. J., Slber, G. R. and R. (1996) 1, I%hann. Sci. 85, 547-553 E. Y. rtal. (1997) Adv. Nc~rml. 72, 121-132 R. Y. and Ma&s, J. D. (1996) Clrn. &.trosli. 3, 310-316 U., N&&in, L., Vetma, I. M., Trono, D. and Gage, F. H.
47 Blamer, (1996) HLtm. Mol.
Genet. 5, 1397-1404
Emerging tandem-massspectrometry 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,ighthroughput 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 the field of biological mass spectrometry. The inception of such ‘soft’ ionisation techniques as fast-atom bombardmentlJ, electrospray3 and matrix-assisted laser desorption ionisation (~JIALDI)~*~ in the mid1980s made it feasible to ionise and introduce into the gas phase thermally labile compounds such as proteins, peptides, carbohydrates and oligonucleotides. These advances in ionisation methods, coupled with various mass analysers, have generated new, technologically sophisticated, mass spectrometry instrumentation that is currently being used to characterise the primary structures of peptides and proteins involved in complex A. R. Don@ ([email protected]}, J. K. Eng (eqj@ u.washington.edu) and J. R. Yates III [email protected]~~tor2.edu) are af the Deparrmetit of A4okrulav Biotechnology, University of Washington, Seattle, WA 98195, USA. A. R. Dar@ is also al the Department ofImmunology and the Howard Hughes Medical Institute al the University of Washiqgton, Seattle, WA 98195, USA. TIBTECH OCTOBER 1997 NOL 15)
Copynght
Q 15197, tlsevler
Sctence
biological processes. These processes, such as antigen processing and presentation, covalent modifications associated with signal transduction and single point mutations, can now be readily studied using tandem mass spectrometry7-“. The worltdwide large-scale DNA sequencing efforts that are currently being pursued will further aid in the advancement of mass spectrometry to study the complex biological processeslO. The complete genomic analyses of organisms such as Saccharomyces cevevisiae and Haemophilus influenzae, Escherichia coli, and the impending complete sequencing of the human genome, will create rich genomesequence databases 11 that will enhance our ability to study both physiological and biochemical processes. The primary goal of a biological study is to identify the functional species involved in a process, and involves placing a biological context on genes and gene products. Tandem mass spectrometry is ideally suited to provide a powerful link between genomics Ltd. All rights reserved.
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HI: SO167-7799(97)0111@l
reviews
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 neurodegenerative
of the mechanisms
disorders
involved in the development
and the actions of neuroactive
of
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 from the lack of simple hormones or peptides, the administration of these compounds in a controlled fashion could provide therapy. Conditions such as diabetic neuropathy, amyotrophic lateral sclerosis (ALS) and Huntington’s disease, and possibly also Alzheimer’s and Parkinson’s diseases, could benefit from this type of approach. Nevertheless, although some of the candidate compounds for treatment have been identified, an adequate means of long-term delivery to the nervous system is still lacking. Only a limited number of small, stable molecules acting on neurons or nonneuronal tissue are available for long-term treatment that can be administered orally. However, peptide drugs and small peptides are easily degraded by proteolytic enzymes and hence cannot reach their site of action when administered in this way. The strategy for the administration of these readily hydrolysable molecules for the treatment of neurodegenerative conditions of the central and peripheral nervous system (CNS and PNS, respectively) can assume two directions - either the defective or absent gene is replaced, with the expectation that the missing compound will then be produced, or the missing compound is provided directly. This article focuses on the latter approach, which can be achieved by direct delivery of the molecule or through the use of living cells that produce the missing substance. D. Maysinger [[email protected]) and A. Morinville are at the Department $Pharmacology and 7’herapeutics, McGill University, 3655 Drummond Street, Room 13 14, Montreal, Quebec, Canada H3G lY6. TIBTECH OCTOBER 1997
(VOL 15)
Copyright
0 1997,
Elsevier
Scmce
Drug-delivery systems ‘Microcapsules’ contain an agent in their centre surrounded by a polymer or a macromolecular carrier. A drug (or other active agent, or a living cell) is in the core of the microcapsule and the surrounding polymer layer can vary in thickness and permeability, and their diameter usually ranges between 1 and 2000 micrometresi. Microcapsules are also known as microspherules, spansules, and coate’d granules, pellets or seeds’, and can have several separate cores surrounded by a polymer or a mixture of polymers. ‘Microparticles’ are matrices containing drugs (or other agents) more or less homogeneously distributed (suspended) throughout the entire matrix with no distinct core or envelope. Their dimensions are in the micrometre to nanometre range. ‘Microspheres’ (nanospheres) represent examples of microparticles (nanoparticles). Examples of microparticles and microcapsules and other delivery devices containing neuroactive agents or living cells for administration to tlhe CNS and PNS are provided in Table 1. Targeted drug delivery and the related problems Two main categories of targeting exist - passive and active. Passive targeting occurs because of the body’s natural responses to foreign material; macrophages from the liver and the spleen play the pivotal role in these actions. Small particles are not as subject to reticuloendothelial-system (RES) uptake as larger ones, and polymeric materials can also influence this process - for example, hydrophilic, neutral surfaces are favoured over more lipophilic larger particles. In Ltd. All rights reserved.
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reviews contrast, active targeting may involve the attachment of a receptor device or vector molecule to the drugdelivery system. If the target site is the liver or spleen, the drug-delivery vehicle should be designed to activate the complement system in order to promote RES uptake. If the target is another organ, the vehicle must avoid RES uptake and should include a vector molecule that will direct it to this organ. However, this task remains difficult and only limited success has been achieved so far.
Liposomes
Drug-delivery devices Microcapsules Microsphere-matrix type Liposomes Micelles Conjugates
Gelforms Collagengels Osmoticmini-pumps Adhesivepatches
Living-cell-delivery devices
Since their discovery by A. D. Bangham in the 196Os’, liposomes have received much attention as drug carriers. Conventional liposomes resemble plasma membranes, consisting of phospholipid molecules with a polar head with two hydrophobic tails, forming a bilayerj. In contrast, micellar subunits possess one such tail and form a single layer3. Liposomes are biocompatible, nontoxic and biodegradable2J,“, and offer the possibility of carrying hydrophobic, hydrophilic or amphiphilic molecules3. Despite these numerous attributes, liposomes have not gained widespread use as drug-delivery systems, due to their instability in viv$J,“. Classical liposome preparations, sterically stabilized liposomes and immunoliposomes are all susceptible to clearance by the immune system’J,j. Most work on liposomal applications has centred on the delivery of anticancer agents and has culminated in the liposomal formulation of doxorubicin3.5. Recent advances in liposomal formulations include cationic liposomes used to entrap various polynucleotide@. In addition, cationic liposomes have been complexed with DNA; these are synthetically based nonviral carriers of DNA vectors for gene therapy”, but the in vim lifetime of these promising formulations remains to be determined. At present, the working in vivo range of liposomes is restricted to the nanometre scale2 as long-term circulation depends on size: the larger the liposome, the faster the clearance. Because the action of neuroactive agents (for example, neurotrophic factors) is mostly achieved by their coupling to a receptor on the plasma membrane, causing an intracellular cascade of events, the formulation of these molecules for delivery must allow sustained extracellular release. Sterically stabilized liposomes, which are taken up by endocytosiss, are therefore unsuitable for this purpose. Finally, owing to the often potent nature of neuroactive agents, sustained drug release, entailing extended in vim stability of the delivery system, remains difficult with liposomes. In conclusion, liposomes are presently unsuitable for the long-term administration of drugs to the CNS and PNS unless adequately stabilized. Microcapsules and microparticles A delivery device for long-term treatment nervous system needs to possess a lifetime that that of the entity it is carrying, which may example, a peptide molecule or a living cell. the difficulties in penetrating the blood-brain
Table 1. Delivery of drugs and cells to the central and peripheral nervous systems
of the exceeds be, for Due to barrier,
Encapsulatedcells Injectionof dispersedcells
Hollowfibres Collagengels
research into the long-term administration of drugs to the CNS has classically been directed at permanently installed devices. In order to avoid inconvenient inse:rtions and the possible removal of large implants or permanently installed stainless-steel cannulae, several types of injectable drug formulations, including microcap sules and microparticles, have been applied in neuroscientific research, showing some promise for clinical use. On the other hand, systemic or local drug delivery is needed for administration to the PNS, for example in diabetic neuropathy. Regardless of the ultimate goal, one of the major obstacles remains the choice of construction materials; the biocompatibility and controlled rate of drug release of the material are impo:rtant variables in the selection process. Table 2 lists some examples of materials used for the encapsulation of neuroactive agents. Polymers that can be used for the preparation of implants or microspheres may be either biodegradable or nonbiodegradable, with both types having eno-rmous potential for drug delivery in the CNS and for use as vehicles for transplanting viable cells7.8. Polysulfones’, poly(acrylonitrile-co-vinyl chloride)*O, ethylene-vinyl acetate (EVA) and hydroxyethylmethacrylate-methyl-methacrylate copolymer (HEMA-MMA)7,8 are frequently used nonbiodegratlable materials. These water-insoluble systems yield capsules that are mechanically and chemically stable and are convenient for cell implantation’. The pol;rmer acts as a semipermeable membrane, allowing the diffusion of the neuroactive agent. However, encapsulation of living cells using these types of materials pnsents numerous difficulties because of toxic effects of solvents on cells, either during the formation of thermoplastic-based capsules or because of residual solvent. Biodegradable polymers can be either synthetic or natural and may be degraded in vivo both enzymatitally and nonenzymatically. Their byproducts should be biocompatible, nontoxic and readily excreted. Only a few immunologically tolerable biodegradable polymers are available. Human serum albumin and bovine serum albumin have been extensively used for target-’ ing anticancer drugs, insulin, and monosialoganglioside TIBTECH OCTOBER 1997 WOL 151
412
reviews Table 2. Examples of materials used for encapsulation7.9JOJ3 Name
Source
Biodegradability
Comments
Humanserumalbumin, bovineserumalbumin Collagen,haemoglobin, gelatine,chitosan,alginate andpoly-L-lysine PolyOactide-co-glycolide)
Natural
Biodegradable
Natural
Biodegradable
Nontoxic, immunologically tolerable Nontoxic, immunologically tolerable,doubtfulpurity
Synthetic
Biodegradable
Polyhydroxyalkanoate
Naturalor synthetic Biodegradable
Ethyl-vinylacetate, Synthetic hydroxyethyl-methacrylatemethyl-methacrylate copolymer Polysulfones, Synthetic polyfacrylonitrile-co-vinyl chloride) Fumaricandsebacicacids Synthetic
Nonbiodegradable
Versatile,controlleddrug release, erodable Thermoplastic,widevariety of repeatingsubunits,controlled drugrelease Thermoplastic
Nonbiodegradable
Retrievalafter implantation
Biodegradable
Adhesivemolecules,increased in viva lifetime,usefulfor drug deliveryto gastrointestinaltract
(GMl). Other natural biopolymers, such as collagen, haemoglobin, gelatine, chitosan, alginate and poly-Llysine, have been employed as carriers for parenteral administration’t.la.13, but their cost and lack of purity have hindered their use. Therefore, several types of synthetic biodegradable polymers have recently been developed and used for microencapsulation, because their processing conditions, availability and cost can be controlled more efficiently’. Among the biodegradable polyesters, which hold considerable promise for the controlled delivery of proteins, are those based on poly(lactic acid) and poly(glycolic acid). Poly(lactide-co-glycolide) (PLGA), commonly used in sutures, provides great flexibility in terms of composition, thus allowing tailored drug release. The biodegradation and tissue reactions of PLGA have been extensively investigatedr”,ls. PLGA is a linear polyester prepared by ring-opening polymerization and subsequently purified to yield a with the chemical structure polymeric powder [-H2C-CO-O-],, [-CH(CH3)-CO-O-],,, and it hydrolyses by an acid- or base-catalysed reaction to form lactic and glycolic acids. The degradation properties of PLGA depend on the molar ratios of the two monomers in the polymer chain, the molecular mass, crystallinity, size and shape of the device, and on the implantation site 16. For example, the time taken for PLGA copolymer to biodegrade can be varied from about 30 days to almost a year1ss17 by adjusting the lactic acid:glycolic acid ratio. Polyhydroxyalkanoates (PHA) represent another class of biodegradable materials amenable for microencapsulation and are potentially superior to PLGA. These biodegradable plastics are synthesized by a number of bacterial species’s,‘“. The majority of PHAs contain from three to six carbon atoms in the polyTIBTECH OCTOBER 1997
(VOL 151
ester backbone, with numerous va:riations in the Rpendant chain’*. Most poly-R-(-)-Shydroxyalkanoic acids are composed of a three-carbon-atom backbone and up to eleven carbon atoms in the pendant chainIs. The structure for these poly-R-(-)-3-hydroxyalkanoic acidslsJ” is [-0-CHR-CH,-CO-O-],, where R represents the pendant chain. This variation in repeating units confers a greater flexibility for tailoring the agent-release properties compared to PLGAs. Because they are part of the natural biosynthesisbiodegradation cycle, PHAs have few or no side effects and, under aerobic conditions, they degrade to water and carbon dioxide’“. The most widely used of these thermoplastic esters is poly(3-hydroxybutyrate), commonly abbreviated to P(3-HB) or PHB. PHB is often employed in conjunction with hydroxyvalerate to form copolymers such as poly(3hydroxybutyrate)(3-hydroxyvalerate), P(3HB-3HV). PHAs have been employed to microencapsulate, among other things, anticancer drugs such as l-(2 chloroethyl)-3cyclohexylnitrosourea (CCNU)20 and 2’,3’-diacyl-5fluoro-2’-deoxyuridine. Block-copolymer micelles in drug delivery Traditionally, micellar drug-delivery systems have been formed from small-molecule surfactants and amphiphilic block-copolymer molecules. The incorporation of the drug into small-molecule surfactant micelles is quite low, representing one of their major limitations as successful delivery systems. In contrast, block-copolymer micelles can incorporate agents to a greater degree. Block-copolymer micelles are formed from individual block-copolymer chains containing a hydrophobic block and a hydrophihc block. A variety of shapes and sizes (between 10 and 100 nm) can be produced using technologies developed by Eisenberg
413
reviews
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 favourable for avoiding capture by the RES. The amphiphilic character of the block copolymers enables selfassembly of nanometer-scale aggregates with a hydrophobic core and a hydrophilic outer shell in aqueous media. The hydrophobic core accommodates nonpolar drugs and the properties of the hydrophilic shell can be adjusted to both maximize biocompatibility and avoid RES uptake. Three main groups ofblock-copolymer delivery systems exist - polymeric drugs, micellar microcontainers and block-ionomer complexes21. Polymeric drugs are block-copolymer-drug conjugates in which the drug is covalently bound to the polymer carrier. Common synthetic polymers and copolymers employed as carriers include poly(divinyl-ether-comaleic anhydride), poly(styrene-co-maleic anhydride) and homopolymers such as poly(L-lysine), poly(ethylene glycol) (PEG) and poly(L-aspartic acid). PEGpolyaspartic-acid block copolymers were used to bind adriamycine, and this system showed several advantages over adriamycine in non-micellar formulation. The major advantages were reduced binding to albumin, greater stability in vitro, lower systemic toxicity and apparent higher effectiveness as an anticancer
agent. However, the decreased cytotoxicity was caused by the slower release .of the covalently bound adriamycine from the polymeric Carrie?. Recently, the focus has been on micellar systems including noncovalently incorporated drugs, for example micellar microcontainers. These types of micelles are made of diblock and triblock copolymers, and in aqueous solutions they form a water-soluble shell (corona) with a micellar core formed &om the hydrophobic loops of polymer chains. The dibl’ock and triblock copolymer micelles are both therrnodynamically and kinetically stable in aqueous media and are promising drug-delivery vehicles. Block-ionomer complexes are formed from amphiphilic block copolymers with a charged hydrophobic core-forming block. The compound to be incorporated into these micelles must have the opposite charge from the core-forming block. When the block copolymer is mixed with a drug, a complex is formed because of electrostatic interactions between the core-forming block and the drug. This kind of micellar block-ionomer complex has recently been shown to be useful for DNA complexing, allowing a novel approach to DNA delivery consisting of the formation of DNA-block-ionomer complexes. Kabanov TIBTECH OCTOBER 1997
NOL 15)
414
reviews et al.*” demonstrated enhanced stability of 19-mer oligonucleotide incorporated into micelles of poly(ethylene oxide)-b-polyspermine in serum. Such types of ionomer-micelle complexes are potentially useful as delivery systems in gene therapyad. The therapeutic potential of many drugs, particularly those that are highly liposoluble, could be greatly enhanced by an adequate selection of block-copolymer micelles. The progress in polymer chemistry, in particular block copolymers and rationally designed small peptides that mimic the effects of large neurotrophic proteins, may offer new ways to deliver drugs to the PNS and CNS. Biologically adhesive molecules An exciting new range of materials for microspheres are the so-called biologically adhesive molecules. These molecules possess advantages over the other drug-carrier systems, in that they exhibit increased residence time in vioo and, hence, the effects of the drug they carry could be sustained for longer intervals. Traditional approaches to the development of biologically adhesive molecules have centred on the use of hydrophilic polymers and hydrogels containing carboxyl groupsz5. Recently, a rapidly degrading hydrophobic polymer consisting of polyanhydride copolymers of fumaric and sebacic acids (FA:SA) exhibited delayed passage through the gastrointestinal tract when compared with other polymers due to an increase in biological adhesiona5. Poly(FA:SA) 20:80 nanospheres 4ct120 nm in size, fabricated using phase-inversion nanoencapsulation, were detected in the cytoplasm of cells and also inside the Golgi apparatus and secretory vesiclesas. Within the poly(FA:SA) nanospheres, dicumarol, a low molecular weight compound with low solubility and poor bioavailability, displayed increased bioavailability in animals compared with other modes of oral administration’j. Similarly, orally delivered insulin, encapsulated in a 50:50 blend of poly(fumaric anhydride) and PLGA improved the regulation of glucose in rats in comparison with rats fed an insulin in saline solution or a saline solution alone25. These results should, however, be interpreted with caution, as the animals were not diabetic and the usual parenteral administration of insulin was not assessed in comparison with the oral nanosphere formulation. Systemic circulation of these bioadhesive molecules, required for the treatment of many conditions, was not demonstrated. Despite this, these preliminary studies offer exciting possibilities for the oral delivery of a variety of compounds.
Delivery of neuroactive agents ‘Neuroactive agents’ is a broad term, and includes several categories of peptide, protein and small molecules that act on the nervous system. Neurotrophic agents are substances capable of producing reparation, regeneration or protection of damaged cells in the nervous system. ‘Neurotrophic factors’ are endogenously produced peptides that participate in the establishment and maintenance of the neural phenotype development and adulthood. during embryonic TIBTECH OCTOBER 1997 (VOL 15)
‘Neurotrophins’ comprise a restricted group of neurotrophic factors that are structurally related to nerve growth factor (NGF). Strategiesfor delivery Various approaches have been developed to provide continuous delivery of neuroactive agents, and, although these have overcome some of the problems of delivering these agents, numerous additional problems remain. Even though drug delivery to the PNS appears simpler than delivery to the CNS, problems such as the site of administration, linearity of release, biocompatibility of the polymeric material and loading capacity have still to be solved. F’ermeabilizers of the blood-brain barrier, osmotic pumps, slow-release polymeric devices and particles, and igenetically engineered cells represent some of the tested means of neuroactive-agent delivery. In addit:lon, transfecting neural cells in vivu with viral vectors has been extensively investigated and certainly deserves attention (for reviews, see Refs 26-28). Table 3 summarizes some of the advantages and disadvantages of the main categories of nonviral delivery systems. The encapsulation of growth factors and of cells engineered to secrete them is in its infancy; so far, only a limited number have been tested in in uivo models (rodents and primates)y~ay-31. Several recently described examples of neurotrophin administration using microcapsules and microparticles will be summarized here, with an emphasis on those factors claimed to be effective in rescuing or preventing neurodegenerative conditions. Essentially, two main approaches to the delivery of neuroactive agents in encapsulated formulations exist - biodegradable polymeric microparticles and microcapsules, and nonbiodegradable delivery devices such as hollow fibres that accommodate cells synthesizing trophic factors (see Table 1). Delivery of neuroactive agents using microcapsules and microparticles Among the most studied neurotrophins are NGF, brain-derived neurotrophic factor (BDNF), neurotrophins 3 and 4 (NT-3, NT-4) and glial-derived neurotrophic factor (GDNF). The physiological roles of these and related neurotrophic factors in nervoussystem development and degeneration8v32.33, the signaling mechanisms related to them31 and the problems of their delivery to the nervous system7,* have been extensively reviewed. The administration of growth factors such as NGE insulin-like growth factor (IGF) or human growth hormone (hGH) typically poses many problems, owing to limited oral bioavailability and short in uivo lifetimes3s. A number of human trials involving ciliary neurotrophic factor (CNTF) have yielded disappointing results36, often because of serious side effects (toxicity)37; improvements in drug delivery could lower the therapeutic dose and hence reduce these side-effects36. Sustained-release formulations involving microspheres composed of biocompatible and biodegradable polymers could overcome these
415
reviews Table 3. Properties of common nonviral delivery systems for the peripheral and central nervous systems* Nonviral delivery Site of action (central Advantages system or peripheral nervous system)
Disadvantages
Blood-brainbarrier Central permeabilizers
Highsystemicdoses,unlocalized braindelivery
Osmoticpumps
-
Systemicuptake
Centraland peripheral Cerebrospinal-fluid delivery, long-termdelivery
Failurecould leadto potentiallytoxic 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 compounds. Recombinant human CNTF (rhCNTF), which has possible applications in the treatment of ALS, was encapsulated in PLGA-and-chitosan microspheres and also in dry alginate with chitosan using the phase-evaporation method37. Sustained release was achieved in both cases, with a higher rate of release for the alginate-chitosan microspheres (2-l 2 days), whereas with PLGA-chitosan microspheres, drug release was observed up to 24 days37, but this formulation has still to be tested if2 viuo. Colony stimulating factor 1 (CSF-1) has been implicated in the differentiation, proliferation and survival of microglia, but has typically posed delivery problems caused by enzymatic degradation38. Recombinant human CSF-1 (rhCSF-1) was encapsulated in PLGA-chitosan microspheres by the solvent-evaporation method3*. These microspheres potentiated the microglial response to injury; the effect was slightly less when the microspheres were implanted in the peritoneum rather than at the site of lesion3R. Treatment of diabetic neuropathy could benefit from the encapsulation of neuroactive agents. Reduction of IGF activity has been shown in both human and nonhuman models of diabetesj9. The expression of IGF-I is controlled by and mimics that of hGH, decreasing after the third decade in nondiabetic individuals3”13”. Recently, recombinant human growth hormone (rhGH) has been encapsulated in PLGA microspheres using a cryogenic process35. In monkeys, injection of these microspheres resulted in an increase of both rhGH and IGF-I for a period of one month3”, even though daily injections of rhGH failed to elicit the same response35. Therefore, growth factors within microspheres represent an exciting combination for the potential treatment of neurodegenerative conditions of the PNS.
Living cells for drug delivery Many neurodegenerative conditions could pot’entially be treated by the administration of neuroactive agents in a controlled manner through the use of living cells, either wild-type or genetically engineered, that secrete these molecules. However, immortalized cells grafted into the CNS often give rise to tum’our formation in the host tissue’; the use of primary cells is a possible strategy to overcome this problem. Nevertheless, cell transplantation has typically posed problems due to the low availability of human cells and immunorejection 41, leading to lifelong treatments with immunosuppressants. Immunoisolation offers the advantages of reducing immunorejection ;and introducing the possibility of xenograftsJ1. The immunoisolating device must allow the passage of nutrients and oxygen as well as the active compound of interest, and must be biocompatible with both the host and the transplanted cell. Microencapsulated cells may thus become efficient biological pumps, overcoming some of the difficulties associated with the implantation of dispersed cells or repeated injections ofdrugs (Table 3). PC12 pheochromocytoma cells .and fibroblasts are commonly used in these delivery systems. Encapsulated cells have improved survival .and sometimes even produce more neurotrophin t:han when grown as monolayer culturesZ9.30,37. Anchoragedependent cells such as fibroblasts need a support to provide long-term survival, and matrigel, collagen and polyelectrolytes (alginate, chitosan) are commonly used inside the polymer capsules. The use of hollow fibres of nonbiodegradable polymers has the advantage of allowing the device to’ be retracted when it is not needed or when the cells cease to produce the drug, although surgery would be necessary. In some cases, the administration of neurotrophins or neurotrophin mimetics”* in biodegradable TIBTECH OCTOBER 1997
NOL 15)
416
reviews microspheres is advantageous, because the polymer will gradually erode, yielding biocompatible degradation products that can easily be eliminated by the body without the need for surgical removal. Technological approaches to and the current status of nonbiodegradable hollow-fibre encapsulation have been extensively discussed in recent reviewssJ3. Delivery using encapsulated living cells A variety of devices have been developed for the delivery of living cells, including microcapsules, macrocapsules, h o 11ow fibres, tubular membranes, intravascular devices and flat sandwich pouchesAl. The usual construction materials, such as HEMA-MMA or poly-L-lysine with hardened polyanionic gel, are limited in their utility, because various capsule parameters, such as size, mechanical strength, permeability and wall thickness, are not independent of each otherqi. However, a new method, based on sodium alginate, cellulose sulphate, polymethyleneco-guanidine, CaCI, and NaCl, has the advantage of allowing independent modifications to the capsule parameters and has recently been employed to encapsulate rat Langerhans islets”. This formulation, with living cells transplanted into the peritoneal cavity of mice, produced an effect in both chemically induced diabetic rats and in nonobese-diabetic (NOD) females; these cells functioned for four to six months in the chemically induced diabetic rats and for LIP to six months in the NOD females+‘. This type of therapy offers the advantage of controlled secretion in response to metabolic demands and signals, thus mimicking the actions of functional cells, without the usual problems associated with transplantation. Nonetheless, the rat islets failed, owing, not to rupture of the capsules or an immune attack41, but possibly to the low viability of the islet cells themselves, resulting from the insults sustained during their isolation from the pancreas. No treatment is presently available for Huntington’s disease (HD), which is characterized by cognitive and marked behavioural deterioration and motor changeslo. CNTF has been shown in rodent models of HD to prevent the loss of striatal neurons and to shield against behavioural deficienciesiO. Babyhamster-tibroblast cells, genetically engineered to produce human CNTF (hCNTF), were encapsulated in asymmetrical hollow fibres of poly(acrylonitrile-covinyl chloride) and implanted intrastriatally into cynomolgus monkeys; deterioration due to HD was imitated using quinolic acid (QA) injection one week after implantation”‘. The animals were sacrificed three weeks after QA injection and the capsules retrieved prior to death. In Nissl-stained sections of the striaturn, a significant reduction of lesioned areas in the caudate and putamen was observed in the hCNTFtreated monkeys compared with the controls’“. Similarly, significant protection of glutamic acid decarboxylase-, choline acetyl transferaseand NADPH-d-positive neurons was also provided by the hCNTF-producing capsulesr(‘. In addition, atrophy of neurons in layer V of the motor cortex ipsilateral to TIBTECH OCTOBER 1997 NOL 15)
the lesion was reduced in animals with the hCNTF cells’“. The retrieved implants exhibited a marked decrease in the rate ofsecretion of CNTF with respect to the levels measured prior to implantation (12.7 versus 45.1 ng dayi capsule-‘)I”. The reason for the decreased production was not determmed and the state of the retrieved capsules was not asses:sed.Death of the encapsulated cells would lead to decreased production; this could be caused by insufficient nutrient availabilirq an immune response to the cells or a reduction in cell viability due to the encapsulation conditions. Nevertheless, the neuroprotective efIect on the cortical neurons innervating the striatum generated by these encapsulated hCNTF-producing cells provides encouraging results for the treatment of HD and other neurodegenerative diseases, with potential applications in Parkinson’s and Alzheimer’s diseaxs. However, the feasibility and chronic effects of long-term treatment with these CNTF-producing capsules need to be ascertained. In addition, CNTF has recently been employed for the treatment of ALS’. Baby-hamster-kidney cells, genetically engineered to secrete hCNTE were encapsulated in hollow-fibre membranes of poly-ethersulfone and implanted into the lumbar intrathecal space of six patients with an early stage of AL!?. The production of hCNTF was detected in the cerebrospinal fluid of patients for up to 17 weeks’, and the devices were removed 13 or 17 weeks following implantation”. The major side effects associated with systemic administration of hCNTF were not observed with this form of delivery’. However, despite these encouraging results, the disease continued to progress in these patients and long-term studies involving a larger number of patients need to be conducted. Nevertheless, this mode of administration using genetically engineered cells holds promise for the treatment of neurodegenerative conditions. The use of nonbiodegradable hollow fibres, which must be retrieved post-implantation, represents an alternative to microparticles and microcapsules. Cells from the human embryonic kidney cell line 293, which produces rhCNTE were encapsulated in biodegradable alginate-chitosan microspheres using an extrusion method without the use of organic solvents3’ and sustained in ~ifr~) drug delivery \vas achieved. The use of long-lasting but biodegradable microspheres has the advantage over other invasive devices, such as cannulae and hollow fibres, of not requiring retrieval after implantation3’. These initial promising results need to be tested irz vivcj to determine the feasibility of these microspheres for the long-term treatment of neurodegenerative conditions. Recently, LM-10 tibroblast-like cells producing rhCSF-1 have also been encapsulated in alginatechitosan microspheres and then implanted at the site of a unilateral cerebral cortex ischemic injury or in the peritoneal cavity in mice with no endogenous CSF-13s. The microspheres with LR4-10 cells potentiated microglial response to the injury, resulting in significant neuron rescues”. The survival time of the
417
reviews LM-10 cells within the alginate-chitosan microspheres can be controlled by varying the physical properties of the chitosan3*. These results show promise for the administration of cytokines or other neurotrophic factors to the CNS in a sustained-release formulation and for the use of cells producing such factors. Future prospects Despite huge advances in the field of encapsulation of proteins and genetically engineered cells, many problems remain to be solved. In the treatment of neurodegenerative conditions, some of the substances that could provide rescue or treatment have been identified, but no suitable means of their delivery is in routine clinical use. Some of these problems could be circumvented by the use of small cyclic peptides, but even these molecules need to be delivered in a suitable manner’?. In comparison with the more established methods of administering trophic factors, such as repeated injections or via osmotic minipumps, neurotrophin delivery systems based on genetically engineered cells have several advantages. Firstly, neurotrophins stored in a reservoir may be degraded over an extended period of time, whereas cells can be engineered to produce neuroactive agents continually. Secondly, while only a finite amount of neuroactive agent can 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 are only functional for up to two weeks and have to be replaced if the treatment is required for longer periods of time; this results in greater subject morbidity by, for example, increasing the risk of infection. However, cells can produce neuroactive agents for a much longer period of time and are replaced less frequently; moreover, recent developments in molecular biology suggest that we are now in a position to improve the control of cell function and survival. Due to the ability to control the release of agents microencapsulated by judicious choice of the materials of construction, this type of technology has the potential for use in a number of areas that require numerous injections, such as vaccination. Recently, oil-based PLGA microcapsules fabricated by the solventevaporation method have been described that possess kinetic properties that allowed the release of tetanus toxoid after three weeks or seven weeks-l”. The injection of a cocktail of these microspheres could potentially result in a pulsed release of the tetanus toxoid, mimicking the action of the repeated injections presently required to produce antibodies. In viva studies have still to be performed to assess whether the pulsed delivery from the microspheres will offer immunoprotection 44. This type of vaccination has the potential to reduce costs and improve patient compliance. Similar kinds ofbiodegradable microspheres may prove useful for drug delivery to the nervous system. Many techniques using microcapsules and micropartitles have produced sustained drug delivery in tlitro, and a number of formulations have been tested in ho. Long-term trials of the microparticles and micro-
capsules in viva have still to be conducted in many cases in order to determine the feasibility of sustained drug release and possible long-term side-effects. Recent progress in polymer science improving block copolymers and lipophilic, adhesive and bioerodable polymers will enable further development of improved delivery systems useful for numerous medical applications. Some of the problems associated with tissue-specific and site-specific delivery for long-term treatment of neurological disorders could be overcome by administration of multipotent neural progenitors or stem cells. These cells could distribute a therapeutic gene product in a sustained and direct fashion throughout the nervous system. Moreover, they may replace dysfunctional neurons and glia in both a site-specific and global manner, thus offering alternative strategies for treating neurodegenerative disorders45,4h,47. Acknowledgments We wish to thank C. Allen for her contribution t:o the block-copolymer discussion and L. Zhang and A. Eisenberg for providing illustrations of blockcopolymer micelles. References
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Genet. 5, 1397-1404
Emerging tandem-massspectrometry 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,ighthroughput 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 the field of biological mass spectrometry. The inception of such ‘soft’ ionisation techniques as fast-atom bombardmentlJ, electrospray3 and matrix-assisted laser desorption ionisation (~JIALDI)~*~ in the mid1980s made it feasible to ionise and introduce into the gas phase thermally labile compounds such as proteins, peptides, carbohydrates and oligonucleotides. These advances in ionisation methods, coupled with various mass analysers, have generated new, technologically sophisticated, mass spectrometry instrumentation that is currently being used to characterise the primary structures of peptides and proteins involved in complex A. R. Don@ ([email protected]}, J. K. Eng (eqj@ u.washington.edu) and J. R. Yates III [email protected]~~tor2.edu) are af the Deparrmetit of A4okrulav Biotechnology, University of Washington, Seattle, WA 98195, USA. A. R. Dar@ is also al the Department ofImmunology and the Howard Hughes Medical Institute al the University of Washiqgton, Seattle, WA 98195, USA. TIBTECH OCTOBER 1997 NOL 15)
Copynght
Q 15197, tlsevler
Sctence
biological processes. These processes, such as antigen processing and presentation, covalent modifications associated with signal transduction and single point mutations, can now be readily studied using tandem mass spectrometry7-“. The worltdwide large-scale DNA sequencing efforts that are currently being pursued will further aid in the advancement of mass spectrometry to study the complex biological processeslO. The complete genomic analyses of organisms such as Saccharomyces cevevisiae and Haemophilus influenzae, Escherichia coli, and the impending complete sequencing of the human genome, will create rich genomesequence databases 11 that will enhance our ability to study both physiological and biochemical processes. The primary goal of a biological study is to identify the functional species involved in a process, and involves placing a biological context on genes and gene products. Tandem mass spectrometry is ideally suited to provide a powerful link between genomics Ltd. All rights reserved.
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