A group of researchers based in Scotlandhas recently developed drug delivery vehicles called bilosomes, which are ves-icles prepared from non-ionic surfactantsincorporating bile salts such as deoxy-cholate1. Bilosomes represent a majorstep forward in vaccine technology be-cause they are resistant to disruption bydigestive enzymes and could, therefore,

be used to overcome the many problemsencountered when delivering antigensvia the gastrointestinal tract.

‘Protein antigens are prone to substan-tial degradation by gastric hydrochloricacid and proteolytic enzymes, and gut-associated lymphoid tissue does not absorb antigen efficiently,’ saysJames Alexander (Dept of Immunology,

University of Strathclyde, Glasgow, UK),co-developer of bilosomes. As a result,he explains, larger and more frequentdoses of antigen are required to achievelevels of immunity comparable with those obtained via systemic adminis-tration. This often leads to the inductionof specific systemic tolerance to the antigen.

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Bilosomes: the answer to oral vaccinedelivery?Kathryn Senior, Freelance writer

Figure 1. A schematic representation of the relative stability of bilosomes and conventional liposomes and non-ionic surfactant vesicles(NISVs) in the presence of bile salts. (a) Lipid vesicles are highly susceptible to the detergent effects of intestinal bile salts, which lead tomembrane disruption and vesicle lysis. (b) Lipid vesicles can be formed in the presence of bile salts (e.g. deoxycholate) and this renders themembrane stable to the further effects of intestinal bile salts. The hydrophobic regions of the vesicles are shown in yellow, and thehydrophilic regions are shown in green.

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(a) (b)

Presence of intestinal bile salts

In the face of such problems, why persist in trying to develop oral formu-lations? ‘Most infections – bacterial andviral – initiate infection via mucosal surfaces; therefore, a mucosally adminis-tered vaccine is the most effective wayto induce a protective immune response,’points out co-author James Brewer (Dept of Immunology and Bacteriology,University of Glasgow, Glasgow, UK). Anoral vaccine is generally safer, and alsooffers other major advantages: ‘It can begiven by anyone, not just trained staffwho can give injections, and so is morecost effective and, because people prefertaking a pill to being given an injection,compliance is higher,’ adds Brewer.

Non-ionic surfactant vesiclesPrevious work by Brewer and Alexandershowed that non-ionic surfactant ves-icles (NISV), which are liposome-likestructures that have extremely low toxi-city, can act as adjuvants2. If the spheresare >250 nm in diameter they can induce a predominantly Th1 (cell-medi-ated) immune response3. NISVs can alsoinduce cytotoxic T-cell responses againstprotein antigens4.

‘We decided to follow up several yearsof research in this area by looking at theability of NISV to act as mucosal vaccinecarriers following oral administration,’says Brewer. However, the group had experience of the disastrous effects ofdetergents on NISVs and had observedthat the greatest obstacle to the stabilityof NISVs in the gut was the detergent effects of bile salts. To circumvent thisproblem, the group made bile salts anintegral part of the NISV membrane ‘be-cause it has been recognized for sometime that this stabilizes the lipid vesiclesagainst the action of bile salts in the gut,’explains Brewer5 (Fig. 1).

Efficacy of bilosomesIn the initial phase of the study, the abil-ity of bilosomes to withstand disruptionby bile salts was confirmed by compar-ing the amount of entrapped bovine

serum albumin (BSA) retained in NISVswith that in bilosomes after the additionof bile salt solutions of various concen-trations. Both types of vesicle remainedstable when incubated with a 5 mM bilesalt solution, but when the concentrationwas increased to 20 mM, 60% of the BSAwas lost from the NISVs, compared withonly 15% from the bilosomes. Bilosomes,into which a synthetic measles peptideor influenza subunit vaccine had beenincorporated, were then used in variousimmunization protocols in mice. ‘Themost effective of these was to give twodoses of vaccine, three days apart, andthen repeat this protocol two weekslater,’ reports Brewer.

After immunization, the mice mounteda specific cell-mediated immune re-sponse, as measured by splenocyte proliferation and interleukin-2 (IL-2) pro-duction. Furthermore, the observed anti-body response to the influenza subunitvaccine was equal to that induced by aparenterally administered vaccine con-taining the same amount of antigen. TheTh1:Th2 balance of the response wasalso similar, irrespective of whether the animals were immunized orally withbilosomes, or parenterally with the traditional vaccine form1.

The study appears to confirm that theeffectiveness of bilosomes is a result ofthe preparation of the vesicles with bilesalts. ‘Bile salts have been used as phar-maceutical penetration enhancers fordrugs given orally, and this ability isthought to be because of the membranedestabilizing activity of bile salts,’ saysBrewer. Although bile salts act initially todestabilize liposome membranes, theyalso subsequently stabilize lipid vesiclesagainst the further effects of bile acids.‘So, whereas NISVs tend to be brokendown in the gut, bilosomes are pro-tected,’ he adds. This also seems to holdtrue in vivo; Brewer reports that greateramounts of bilosome-entrapped radio-labelled antigens are retained in the mousegut for longer periods compared withfree antigens (unpublished observation).

Future studiesBrewer and colleagues have done somefollow-up experiments examining thedistribution of antigens prepared in bilo-somes after oral delivery, and have beenworking with bilosome-based vaccines inan infectious disease model. Recently,the international technology commer-cialization company, BTG (London, UK),acquired the rights to bilosome technol-ogy and is currently in discussion withseveral biotechnology companies inter-ested in vaccine development or drugdelivery. Lorraine Jones, a LicencingExecutive at BTG, says, ‘At this stage, it isnot possible to speculate what projectsmight arise, but the first licence agree-ments could be signed in the next 4–9months.’

Brewer is hopeful that bilosomes willbe developed as an oral vaccine deliverysystem for clinical use in the relativelynear future. ‘The only orally adminis-tered vaccine at present is the attenu-ated polio vaccine, which has the prob-lem that it can revert to virulence,’ hesays, ‘Bilosomes are much safer than attenuated vaccines because they haveno potential for reversion and have ex-tremely low toxicity; we hope they willgo far!’

References1 Conacher, M. et al. (2001) Oral

immunisation with peptide and proteinantigens by formulation in lipid vesiclesincorporating bile salts (bilosomes). Vaccine19, 2965–2974

2 Brewer, J.M. et al. (1994) Non-ionic surfactantvesicles as vaccine delivery systems. Instituteof Chemical Engineering: Second Conference onIndustrial Immunology, 4–5 July 1994, Brighton,UK, Book of Abstracts, 34–36

3 Brewer, J.M. et al. (1998) Lipid vesicle sizedetermines the Th1 or Th2 response toentrapped antigen. J. Immunol. 161,4000–4007

4 Brewer, J.M. and Alexander, J. (1992) Theadjuvant activity of non-ionic surfactantvesicles (niosomes) on the BALB/c humoralresponse to bovine serum albumin.Immunology 75, 570–575

5 Schubert, R. et al. (1983) Studies on themechanism of bile-salt-induced liposomalmembrane damage. Digestion 28, 181–190

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