Aprimary task in formulating drug-delivery systems is to package thedrug molecules at high concentra-

tion, and release them later according to apredetermined temporal and spatial pro-gramme. In the body, this task is regularlycarried out by secretory granules, and onpage 459 of this issue1 Needham and col-leagues describe how they have learned fromthat example. By a clever combination ofpolymer-gel science and lipid chemistry,they have constructed a granule mimic thatcan store a drug for a desired period andrapidly release it when properly stimulated.

Hydrogels (crosslinked polymer net-works which, in the right conditions, swell inwater) have long been studied and employedas drug-delivery vehicles2. By physicallytrapping a drug in a hydrogel and using thesieving properties of the matrix to retardmolecular movement, or simply by main-taining the gel in a dry state, the drug can beretained inside the device for long periods.Covalent or noncovalent bonding of drugsto side-chains of the polymer gel is anotherway of storing a drug and releasing it undercontrolled conditions. Researchers havesought ways to effect release and modulaterelease rates by using changes in pH or tem-

perature to alter the degree and rate of gelswelling; similarly, external stimuli canbreak bonds between drug and polymer,allowing the drug to be released. Conven-tional hydrogel-based delivery systemsrelease their contents over periods of hoursto days, but this timescale can be reduced bymaking the gels smaller. In most cases thetime required for complete swelling andrelease of the gel contents varies linearly toquadratically with gel diameter, dependingon the rate-controlling process2,3. So a tenfold reduction in diameter can lead to up to a hundredfold increase in speed.

Although most hydrogel drug-deliveryconstructs are made with artificial polymersor reconstituted polysaccharides or proteins,it has not escaped notice that nature itself hasevolved a very efficient molecular storageand delivery device — the secretory granule4.For example, mast cells can store histamine athigh concentrations for indefinite periods ingranules, and release it within millisecondsafter the appropriate electrochemical stimu-lation. The structure of the mast-cell secreto-ry granule — a dense network of sulphatedproteoglycans, around a micrometre in size— allows it to condense in the presence ofpositively charged counterions such as

found in at least 29 proteins from yeast,worms, plants, insects and mammals. It isthe first protein domain known to bind thisinositol lipid.

The FYVE domain is about 70 aminoacids long, has eight potential Zn2+-coordi-nating cysteine residues, and binds two mol-ecules of zinc. It was originally identifiedafter observations that wortmannin — aninhibitor of many PI3Ks — caused the redis-tribution of EEA1 from the endosome to thecytosol5,6. The FYVE motif is located at thecarboxy terminus of EEA1, and it is homolo-gous to domains found in Vps27p, Fab1p,Vac1p (yeast proteins implicated in vesiculartransport)7–10, Hrs (a mammalian endo-somal protein)11 and Hrs-2 (another mammalian protein involved in calcium-regulated exocytosis)12.

Gaullier et al.2 and Patki et al.3 tie thesefindings together and show that EEA1 bindsto PtdIns(3)P through its FYVE domain.They find that both wortmannin and muta-tions in the FYVE domain that disrupt bind-ing to PtdIns(3)P cause dissociation andredistribution of EEA1 to the cytosol. Burdand Emr4 further show that Vps27p, Fab1pand Vac1p bind PtdIns(3)P in vitro. And herecomes the surprise — Simonsen et al.1 showthat the requirement for PtdIns(3)P can bebypassed if there is an excess of membrane-bound Rab5–GTP. It turns out that EEA1 notonly binds PtdIns(3)P through the FYVEfinger domain, but that it also interacts withRab5–GTP through an adjacent domain inits carboxy terminus (Fig. 2). Thus, underphysiological conditions, EEA1 is main-tained at the membranes by Rab5–GTP andPtdIns(3)P.

The requirement for EEA1 in endocyticfusion has been shown by Mills et al.13 inanother recent paper. If they depleted EEA1from membranes and cytosol, they foundthat fusion was blocked. Moreover, whenthey added the carboxy-terminal domain ofEEA1 only (residues 1,098–1,411), theyobserved the same effect. These residuesencompass the FYVE domain, as well as thesite for Rab5 binding (Fig. 2).

The observation that a single protein(EEA1) can bind both lipid (PtdIns(3)P) and protein (Rab5) provides a unique

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NATURE | VOL 394 | 30 JULY 1998 427

method of targeting to endosomes only.Other organelles, such as the trans-Golginetwork, contain PtdIns(3)P. But, becauseRab5 is found only in the early endosome,EEA1 cannot bind these organelles. So, EEA1provides directionality to Rab5-dependentendocytic fusion. Rab proteins are involvedin many other fusion reactions; for example,Rab3 functions in exocytosis, where an Hrsisoform (Hrs-2) is also involved12. Moreover,Rab6 and p62, a PtdIns3K11,14, are requiredfor budding of exocytic vesicles from thetrans-Golgi network in mammalian cells.This concept of using a protein with dualbinding specificity is an elegant device forintroducing specificity and directionality to

Figure 2 Model for theassociation of EEA1with the endosomalmembrane, based onthe results of fournew studies1–4. Thecarboxy terminus ofEEA1 containsadjacent binding sitesfor Rab5–GTP(amino-acid residues1,277–1,348) andPtdIns(3)P (residues1,349–1,411). ��

��Endosomal membrane

Rab5-GTP

PtdIns(3)P

EEA1 (carboxy terminus)

P

FYVE finger

GTP

different membrane-transport processes.What is more, other membrane-transportevents that rely on FYVE-domain proteinswill probably turn out to have similar depen-dencies on PtdIns(3)P and a small, specificGTP-binding protein.Claudia Wiedemann and Shamshad Cockcroft are inthe Department of Physiology, Rockefeller Building,University College, London WC1E 6JJ, UK.e-mails: c.wiedemann@ucl.ac.uks.cockcroft@ucl.ac.uk

1. Simonsen, A. et al. Nature 394, 494–498 (1998).

2. Gaullier, J.-M. et al. Nature 394, 432–433 (1998).

3. Patki, V., Lawe, D. C., Corvera, S., Virbasius, J. V. & Chawla, A.

Nature 394, 433–434 (1998).

4. Burd, C. G. & Emr, S. D. Mol. Cell 2, 157–162 (1998).

5. Stenmark, H., Aasland, R., Toh, B. & D’Arrigo, A. J. Biol. Chem.

271, 24048–24054 (1996).

6. Patki, V. et al. Proc. Natl Acad. Sci. USA 94, 7326–7330 (1997).

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131, 603–617 (1995).

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J. Cell Biol. 119, 1469–1479 (1992).

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Nature 385, 826–829 (1997).

12. Jones, S. M., Crosby, J. R., Salamero, J. & Howell, K. E.

J. Cell Biol. 122, 775–788 (1993).

13.Mills, I. G., Jones, A. T. & Clague, M. J. Curr. Biol. 8, 881–884

(1998).

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J. Biol. Chem. 272, 20538–20544 (1997).

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Drug delivery

A lesson from secretory granulesRonald A. Siegel

© 1998 Macmillan Magazines Ltd

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428 NATURE | VOL 394 | 30 JULY 1998

Daedalus

A chance of justiceA legal verdict, says Daedalus, pretends tobe a certainty; but in truth it is merely aprobability. Consider, for example, thoseplaintiffs who claim to have been made illby silicone breast-implants, radioactivityor tobacco. Clearly it is impossible to besure that they would not have got just as ill if they had never encountered theseperils.

So Daedalus is musing on the characterand process of a truly scientific court. If,for example, epidemiological evidenceshows that passive smoking in theworkplace increases your chance of gettinglung cancer by 1%, then the judge coulddecide the case won if the number 100turns up on his random number generator,but lost if 1–99 turn up. Chancy orvexatious litigants would be stronglydiscouraged.

It might be fairer, however, to allow anopenly statistical verdict. A defendantfound ‘probably guilty’ (perhaps set at91.7% if 11 jurors out of 12 reckon he didit) might incur a fine or punishmentreduced in proportion. A defendant found‘probably innocent’ (say, 8.3% or 16.7%)might leave the court unpunished, butwith a definite ‘stain’ on his character.Thereafter, until the stain was declaredspent, or expunged by good behaviour, hewould be literally ‘a suspicious character’— which would tell against him if he cameup again on a similar charge.

This approach would fit well intoBritish society. British motorists alreadyaccumulate ‘stains’ on their drivinglicences for each small offence. Enoughstaining cancels the licence. The Britishhonours system awards a ‘shine’ on thecharacter of good eggs and praiseworthytypes; if they later go to the bad, the shinecan be withdrawn again. Affirmativeaction gives whole groups a collectiveshine, entitling them to jobs, presumptionsof virtue or innocence, and so on.

Such an open system of honours anddishonours is much fairer than the ‘dossiersocieties’ run by dictators. Secret dossiers,for some reason possibly connected withthe second law of thermodynamics, onlyaccumulate evidence against their subjects.Only those faceless apparatchiks whonever put a foot wrong or do anythingoriginal, flourish under them. Ominously,dossiers are now growing fast even in thedemocracies, in the form of credit ratings,referees’ reports, compulsory secretreporting of ‘suspicious’ bank deposits,and so on. An open, numerical ‘stains andshines’ system might just stop the rot.David Jones

histamine, serotonin and calcium. These ionsbind tightly to and neutralize the fixed chargegroups, causing the granule network toshrink. Shrinking is favoured when the coun-terion is polyvalent or partially hydrophobic.

If a secretory granule consisted only of a charged network, it would not hold its payload very long in the intracellular or the extracellular environment. Sodium andpotassium ions would invade the granulesand rapidly ‘exchange out’ the incorporatedspecies. Because these metal ions do not bindto the fixed negative-charge groups, theywould establish an osmotic pressure insidethe granule, causing it to swell and release itscontents. To prevent this, the granule is coat-ed with a lipid membrane that blocks iontransport. Secretion of the stored contentsrequires electrochemically stimulated fusionof the granule’s membrane with the cellmembrane, which exposes the matrix to the extracellular medium, triggering ionexchange and swelling.

If nature can do this, why can’t we? Thatwas the question asked by Needham and col-leagues, and their response is outlined in Fig.2 of the paper on page 459. Using technologythat had already been developed5, they syn-thesized crosslinked polymethacrylic-acidmicrogels that had a swollen diameter of 6.5m m at pH 7.4. They then incorporated thehydrophobic, cationic, anticancer drug dox-orubicin hydrochloride at pH 5.0, whichneutralized the acid groups, causing the gelto shrink. As a final step, they used a newlydevised process to coat the collapsed gel witha lipid bilayer, and showed that the lipid was present exclusively at the surface. Thelipid coating all but prevents dissipation ofpH gradients, and the acidified constructremains stable in pH 7.4 phosphate-bufferedsaline, a proxy for body fluids, for at least 48hours. By this means they mimic the storageaspect of a granule.

To demonstrate ‘quick release’, theauthors exposed microspheres in phos-phate-buffered saline at pH 7.4 to electro-poration fields that were strong enough tobreach the lipid coatings. A brief electro-poration pulse led to rapid swelling of thegels which was complete within seconds, and all the incorporated doxorubicin wasreleased within minutes. Although theseprocesses take somewhat longer than they doin nature, the times concerned are more thanacceptable for many drug-release purposes.

How might such a system be used in prac-tice? The particles are of such size that they willbe rapidly cleared by the reticuloendothelialsystem, which consists of scavenger cells thatcontinually patrol the body. So, without mod-ification, the system is probably best suited for local administration, be it subcutaneous,intramuscular or intraperitoneal. Theauthors suggest, however, that the synthesiscan be altered to make much smaller particlesthat will avoid the reticuloendothelial system

and can also leak through the porous capillarywalls of tumours, providing targeted deliveryof anticancer agents. The practicality of thesystem might be further improved by tether-ing certain molecules, ones which bind specif-ically or nonspecifically to target cells or extra-cellular matrix, to the lipid coating, thus local-izing the microspheres at a particular site andperhaps preventing side-effects. Collagen,peptides containing arginine–glycine–aspar-tate sequences and Fab fragments of antibod-ies are logical candidates. This strategy hasbeen investigated with liposomes, anothertype of drug carrier6.

Needham and colleagues’ prototype sys-tem triggers drug release by electroporation,which may be difficult to effect in situ, andthe authors point out that modifications tothe lipid coating may make it sensitive tostimuli such as temperature or ultrasound.Work with liposomes also points to a second,potentially powerful, refinement of the system7,8. By anchoring suitable stimulus-sensitive polymers in the membrane, one candestabilize the lipid coating, and thereforetrigger drug release, by changes in local tem-perature, pH and glucose concentration, orby illumination at a particular wavelength.Even more sophisticated systems can beenvisaged in which lipid-coated, bioadhesivemicrogels that respond to different stimuli,each type of microgel containing a differentagent, are mixed together and deliveredlocally or regionally. Such a system couldpermit localized combination therapies, inwhich the delivery of the different agentsoccurs according to a predeterminedsequence.

These kinds of elaborations would addfurther complexity to Needham and col-leagues’ system — the combinatorial possi-bilities are numerous, and would require agreat deal of further development and test-ing. Moreover, the effectiveness of the origi-nal concept needs to be tried out in an animalmodel. At the least, however, the authorshave succeeded in showing how some cunning chemistry can be used to emulate aphysiological process for the purposes ofimproved drug delivery.Ronald A. Siegel is in the Departments ofBiopharmaceutical Sciences and of PharmaceuticalChemistry, School of Pharmacy S-926, University ofCalifornia at San Francisco, San Francisco,California 94143-0446, USA.e-mail: ras@rebus.ucsf.edu

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