also be controlled. ‘We have observedthat the destruction of biocompatiblecapsules by enzymes can be varied fromnearly indestructible to rapidly destroyed,’Voigt continues. ‘It depends on the com-ponents used and the medium condi-tions, like pH, salt, temperature and light.’

If the properties of the drug make itimpossible to encapsulate directly, col-loidal particles or even cells can be usedas templates2 (Fig. 1). Once the poly-electrolyte shell has been constructed,the core template is dissolved away toleave a hollow structure. This can be reversibly opened, for example, by achange in pH, enabling even large drugmolecules to enter. Alternatively, thedrug can be precipitated in by generat-ing nucleation centres in the capsulelumen.

Controlled releaseControlled-release delivery is likely to bean important application. The PEM cap-sules are semi-permeable and the widerange of possible encapsulation materi-als gives plentiful scope for adjustingpermeability properties. Researchers atthe Max-Planck Institute created simpleibuprofen PEM microcapsules usingpolysaccharide layers as the coating3.Ibuprofen has low solubility in water.Drug release was studied in vitro in simu-lated gastric (pH 1.4) and intestinal

(pH 7.4) fluids. The ibuprofen crystalsdissolved and the drug diffused out ofthe intact capsule. The rate of releasewas found to depend on the size of thecrystal, the thickness of the capsule walland the solubility of the drug in thegiven medium. There was no rapid initialburst of drug release or incomplete re-lease of the drug dose from the carrier,both of which are problems that are usually associated with the use of micro-carriers.

Future prospectsVoigt is optimistic about the versatility ofPEM capsules. ‘We have found that allthe limitations we thought would existhave vanished after a few months’ work,’he says. ‘I don’t see any limitation to thetype of molecules we can encapsulate.’However, the technology has not yet been tested in vivo. CapsulutionNanoScience hopes to start animal stud-ies with an encapsulated drug in early2002. Toxicity is not expected to be aproblem because all the polymers usedwill be taken from the generally recog-nized as safe (GRAS) list.

‘I am confident that we will begin tosee several applications of the layer-by-layer coating technology over thenext five years, including drug delivery,’says John Lally, Head of Polymers andInterfaces at Ciba Vision Corporation

(Duluth, GA, USA). ‘Its main advantageis the ease of fabrication and the abilityto incorporate a wide range of entities.However, more work is needed in defin-ing the toxicity profile of the nanocap-sules, especially to determine their fateand distribution in the body through animal studies. In addition, some modeldrugs may need to be studied to demon-strate a tangible benefit for drug deliveryapplications. Depending on the route of administration, the stability of thenanocapsule wall could become anissue’.

The direction of future work will de-pend on client requirements: CapsulutionNanoScience hopes to attract interestfrom companies wishing to formulate a range of new and existing drugs. Thetechnology is also being developed foruse in diagnostics, cosmetics and plantprotection products.

References1 Donath, E. et al. (1998) Novel hollow

polymer shells by colloid-templated assemblyof polyelectrolytes. Angew. Chem. Int. Ed.Engl. 37, 2202–2205

2 Sukhorukov, G.B. et al. (1998) Layer-by-layerself assembly of polyelectrolytes on colloidalparticles. Colloids and Surfaces A:physicochemical and engineering aspects.137, 253–266

3 Qiu, X. et al. (2001) Studies on the drugrelease properties of polysaccharidemultilayers encapsulated ibuprofenmicroparticles. Langmuir 17, 5376–5380

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The first insights into the molecularmechanism underlying the process ofviral budding in HIV-infected cells aresuggesting a whole new range of drugtargets that could prove useful in thesuppression of AIDS in HIV-positive pa-tients. Wesley Sundquist’s group at the

University of Utah School of Medicine(Salt Lake City, UT, USA), working in con-junction with collaborators at MyriadGenetics (Salt Lake City, UT, USA), haverecently revealed that HIV-1 uses cellularmachinery to bud from infected cellsand the protein Tsg101 is an essential

requirement of this process. BlockingTsg101 could, therefore, prevent HIV-1budding.

The life cycle of HIV-1The life cycle of HIV infection is knownto consist of six major events: attachment

Budding new HIV therapies?Kathryn Senior, Freelance writer

of the virus to the host cell; reverse transcription of HIV RNA; integration ofHIV DNA into the host cell; transcriptionof viral proteins; translation and viral assembly; and, finally, budding and maturation. HIV-1 assembly is driven bythe viral Gag protein, which is activelytransported to the cell membrane. Oncethere, it forms enveloped, spherical par-ticles that bud from the cell. The buddingevent itself occurs when the cell mem-brane is broken and then resealed to cre-ate discrete viral and cellular membranes1.

Although the Gag protein is an essen-tial requirement for viral budding, HIV-1does not have the genes to code for proteins to break and reseal the cellmembrane, and it is thought to recruitcellular proteins for this job. A potentialdocking site for these proteins has beenidentified on the Gag protein, in the p6 domain. Mutations within the gaggene at this point allow viral assembly tooccur normally but the cell membranecannot be disrupted, causing abnormal,severely attenuated viral release2.

Looking for a cellular Gag-bindingprotein‘One of the main aims of our researchwas to identify cellular proteins thatcould bind to this area of the p6 domainof the HIV-1 Gag gene – an area termedthe PTAP motif. We did this using ProNet™technology developed by Myriad,’ ex-plains Sundquist. ProNet is a yeast two-hybrid-based system for discoveringnew protein-binding partners for a pro-tein of interest. In initial experimentsperformed at Myriad, HIV-1 p6 was usedas a ‘bait’ to screen for potential bindingpartners in a human spleen cDNA li-brary. Genes encoding nearly full-lengthTsg101 were isolated twice, and this wasthe only gene detected in the screens.‘Subsequent experiments in our labora-tory, that were planned in consultationwith Kenton Zavitz and Scott Morhamfrom Myriad, showed that full-lengthTsg101 bound wild-type p6 in directedtwo-hybrid liquid culture assays. The

same experiment performed with threedifferent p6 mutants, all with differentpoint mutations in the PTAP motif, failedto show binding,’ reports Sundquist3.

A molecular mechanism forbuddingFurther studies showed that depletingTsg101 in the cell arrested HIV-1 matur-ation at a late stage (Fig. 1). ‘We thinkthat Tsg101 can bind to the p6 domainand also to ubiquitin, another protein in-volved in viral budding. One attractivemolecular model put forward by theteam suggests that ubiquitination of HIV-1 Gag during viral assembly createshigh-affinity binding sites that recruitTsg101 to assist in the final stages ofbudding,’ says Sundquist. The choice ofTsg101 by HIV-1 could be explained by the usual role of Tsg101 in a normalcell process called vacuolar protein sorting (Vps). The Vps pathway sortsmembrane-bound proteins for eventualdegradation in the lysosome or vacuole,and involves the breaking and resealingof membranes.

A new drug developmentstrategy?Although these results are exciting, thereis much work still to be done. ‘In myview this is just the beginning,’ stressesSundquist, who adds that the mechanicsof membrane breakage and resealing arenot yet understood. ‘Tsg101 is only partof the machinery that is taken over byHIV-1 – now we need to find the rest ofit and show how it fits together,’ he says.In the meantime, Myriad researchers areinvestigating Tsg101 and other cellularproteins that have been identified as im-portant in viral budding and are activelyseeking to develop drugs that targetthem. ‘We have already constructedhigh-throughput drug screens aroundseveral proteins and some of the ‘hits’have been tested for anti-HIV activity inhuman white blood cells in culture,’ ex-plains Kenton Zavitz, Senior Investigatorat Myriad. Many of these hits show

potent anti-viral activity with no appar-ent toxicity. One, MPI49839, is due toenter pre-clinical safety and evaluation inanimals. ‘This will be done mainly from atoxicological and pharmacokinetic pointof view since there is not a good animalmodel for HIV, so it is not possible to testfor efficacy in animals,’ points out Zavitz.Once these studies have been com-pleted successfully, the first candidatescan enter Phase I clinical trials, but ‘it isdifficult, if not impossible, to predict thetimeline that this research will take,’ hestresses.

Avoiding drug resistanceAll current anti-HIV drugs inhibit the re-verse transcriptase enzyme encoded by

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Figure 1. An electron microscopeimage of HIV particles budding from acell (a). Following prevention of Tsg101production, which is necessary for viralbudding, the HIV particles ‘clump’together in a chain (b), leaving themunable to spread and cause theinfection of neighbouring cells. Figures credited to Uta von Schwedler,University of Utah (UT, USA; http://www.utah.edu/unews/releases/01/oct/aidsbud.html).

HIV genes. The use of protease inhibitors,together with nucleoside analogues, isrelatively successful at reducing the viralload to undetectable levels, but HIV ex-hibits a high level of antigenic variationleading to the increased generation ofdrug-resistant strains. In fact, ∼ 25% ofthe 3000 new cases of HIV diagnosed inthe UK each year are infected by drug-resistant strains. A potential anti-HIV drug

based on the inhibition of Tsg101 andother targets is, therefore, particularly attractive, as Myriad’s president AdrianHobden points out. ‘The search for drugsto block cellular proteins that are takenover by the virus has one potential majoradvantage; the target here is a host protein, not a viral protein, so we hopethat the virus will not find a way to getaround it quite so easily,’ he predicts.

References1 Freed, E.O. (1998) HIV-1 gag proteins: diverse

functions in the virus life cycle. Virology 251,1–15

2 Huang, M. et al. (1995) p6Gag is required forparticle production from full-length humanimmunodeficiency virus type 1 molecularclones expressing protease. J. Virol. 69,6810–6818

3 Garrus, J.R. et al. (2001) Tsg101 and thevacuolar protein sorting pathway areessential for HIV-1 budding. Cell 107, 1–20

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Small pellets of porous silicon have beendeveloped that could make targeteddrug delivery and diagnosis a much simpler process. Implanted under theskin, these pellets could monitor bloodlevels of an administered drug, releasenew doses when necessary and thencompletely dissolve once empty.

pSiMedica (Malvern, UK) was set up as a joint venture in August 2000 by the UK’s Defence Evaluation & ResearchAgency (DERA; Malvern, UK) and SumichGroup (Perth, Australia) and has alreadypatented a form of porous silicon(BioSilicon™), which is both biodegrad-able and biocompatible. The companyhopes this invention could help to solvemany of the existing problems with drugdelivery: these include noncompliance,painful injections and adverse effects re-sulting from the systemic bioavailabilityof the drug.

Leigh Canham, Chief Scientific Officerof pSiMedica, said: ‘We are optimisticthat this type of porous silicon gives us amaterial platform that is very flexible. Wecan tune the rates at which it degradesby altering its microstructure and varyingpore size and pore density. The pore sizedistribution and the porosity that youachieve can be varied by changing an-odization parameters like silicon resistivity,

electrolyte composition and current density.’ He emphasized that, unlike bio-degradable polymers, the chemistry ofporous silicon does not have to be alteredto achieve different rates of degradation– a distinct advantage when it comes toapplying for regulatory approval.

Porous siliconSilicon is, of course, the raw material forthe silicon chip and semiconductors. Itcan be produced to a high level of purityand is relatively cheap: even a highlypure form of silicon (99.99%) costs lessthan US$30 per kilogram. Canham beganstudying silicon while working at DERA,where he was involved in a project to de-velop a silicon laser.

However, after extensive reading aroundthe subject, Canham noted some simi-larities between porous silicon andbioactive ceramics, which were being investigated for orthopaedic applica-tions. This led him to investigate whethersilicon could be biocompatible. To hissurprise, in vitro tests showed that thinlayers of highly porous silicon couldcompletely dissolve away in simulatedbody fluids1. Subsequently, a six-monthstudy using guinea pigs showed thatsolid silicon implants could persist in the body without rejection, whereas

implants of porous silicon continuouslydecreased in weight over the study pe-riod2 (Fig. 1). Canham, together withcollaborators at St Thomas’s Hospital(London, UK), have shown that poroussilicon will break down in the body intothe harmless compound silicic acid,which is present in many foods anddrinks (Canham et al.; unpublished data).

Drug deliverySilicon can be made porous by eitherelectrochemical or chemical etching3

and these techniques can be used forboth silicon wafers and silicon powders.However, Canham pointed out that ‘Formany drug delivery applications, you donot necessarily want chips or segmentsor wafers – you want microparticles ornanoparticles. You can either start with asilicon film, porosify it and process it intoa powder, or you can begin with siliconpowder of the required size and shapeand porosify that. We are investigatingboth options.’ Powdered porosified sili-con could, he said, be incorporated intoordinary capsules for oral delivery, intopatches for transdermal drug delivery orinto microparticles covered with the appropriate antibodies that could be in-jected into the bloodstream, for example,to lodge in tumours.

Holey chips for drug deliverySharon Kingman, Freelance writer