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The renowned immunologist and winner of the Nobel Prize for medicine, Paul Ehrlich, was a visionary, having already imagined at the very start of the 20th century that 'magic bullets' could be used to precisely target specific micro-organisms with toxins without flooding the body. It was to take three-quarters of a century before the first research on liposomes was published. In one fell swoop we were thrust into the future, towards nanotechnology! Significant challenges need to be overcome: an adequate vector has to be produced, the diseased cell has to be targeted and the active ingredient has to be protected, but must be released at the right time on target!

Ehrlich was already aware of the blind strength of some systemically-administered remedies which provoke toxic effects which can sometimes be as significant as the illness they are trying to treat. At this stage, he didn't know about antibiotics, which destroy the commensal flora alongside the pathogenic bacteria, nor anti-cancer chemotherapy which aims to destroy tumour cells, but at the cost of significant collateral damage.

The new science, called 'nanopharmacy' or 'nanomedicine' seeks to fine-tune 'transporters' which are capable of specifically targeting a diseased organ or tumour and, once there, of releasing the active ingredient which they are carrying. In some ways, this is a vital step for future progress: if we want to beat certain cancers by administering increasingly toxic molecules and/or at increasingly strong doses, this can only be possible if we are able to target them with precision. Because the best way to reach them is generally through the blood supply, the ideal would be to enable them to travel 'incognito' and only to activate them once they are in contact with their target.

Another avenue for research which may also benefit from nanopharmacy is gene therapy, because genetic material, as well as peptides or proteins would immediately be damaged in the body if they were injected without protection. It should be noted that gene therapy also uses viral vectors, which are not covered in the context of this article.

'We are thus facing a double challenge in nanopharmacy', states Géraldine Piel, Senior Researcher in the Laboratory of Pharmaceutical Technology of the University of Liège: 'targeting the diseased cell and protecting the active ingredient.' And, as we shall see, it is easier said than done!

Active or passive targeting

Let's start with targeting: it may be active or passive. If the target is identified/identifiable by biochemical or immunological characteristics, we can try to steer the active ingredient specifically towards it, by placing 'ligands' on the surface of the vectors, which can identify them. The target site may be a specific receptor which is over-expressed in the diseased tissue, specific small peptides, or quite simply folic acid (tumours often have several folate receptors). However, all this remains hypothetical: there are currently no vectors on the market which use active targeting.

Passive targeting, however, appears to be closer to the mark. Indeed, even the size of the vector or changes on its surface can be used to reach the target. For example, we know that round an inflamed or tumoral site, the internal lining of the blood vessels (endothelium) changes, presenting fenestrations which are approximately 500 nanometres in diameter. If nanovectors have a smaller diameter (between 100 and 200mm), they can slide into the fenestrations and specifically cluster around these points. Passive targeting simply uses the size of the vector, without specifically and immunologically recognising the target or using a particular target receptor.

Disguising progress

As mentioned above, active ingredients are often biological molecules, which risk being identified by opsonines in the immune system as soon as they enter into the bloodstream, and will thus be destroyed before they reach their target. The most widely used tactic is therefore to 'disguise' them so they can move about unseen.

The first generation vectors were simple liposomes: the active ingredient was encapsulated in a lipid bilayer. But these first generation nanovectors were generally recognised by opsonines as soon as they were injected, and ended up in the liver, where they accumulated. They are useful, therefore, for treating ... the liver. Indeed, they have been used to target hepatic cancers.

To avoid opsonisation, some scientists had the idea of grafting polymer molecules onto the liposomes (polyethlylene-glycol = PEG), which made the liposomes 'hairy', thus disguising their immunological patterns which could be identified by the opsonines. These vectors are today known under the name of PEGylated liposomes. For the record, they are also known as 'stealth liposomes' because they were developed during the Gulf War, when the general public first heard mention of stealth aircraft, hence the nickname. The hairy liposomes were said to have 'prolonged vascular persistence' because they could remain for long periods behind their 'masks' in the general circulatory system, which gave them time to infiltrate the points where the vascular endothelium was festrated, i.e. often around the diseased organ.

'But we realised that protecting the active molecule and transporting it to the place were it has to act was still not enough', continues Piel. 'We thus moved towards complexifying these vectors, with a third generation of liposomes (adding biomolecular recognition markers, such as folic acid, antigens, peptides, etc., which would enable more precise targeting - thus overlapping with active targeting projects) and probably a fourth generation. This fourth generation, as well as involving a complex envelope and active molecule, include molecules which react to a stimulus to release the active ingredient.' This is yet another additional, and unexpected, challenge.

Releasing at the right time

Imagine that the nanovector has arrived intact at the right place: its mission isn't yet over. If the active ingredient which it encapsulates has not been released en route, it now has to be released at the target cell. To do so, the first step is to ensure that it can penetrate the cell, which is most often done through endocytosis, and apparently without too many difficulties.

But then, how is the active ingredient released? Different tricks are used to attempt to make the capsule react to stimuli which it will only encounter at the intracellullar level. 'For example,' explains Piel, 'as we know that the pH is more acidic at the intracellular level, and in particular in tumoral cells, we may choose components which change structure when the pH becomes more acidic to encapsulate the active ingredient, meaning that the vesicle bursts. '

External stimuli can also be used, for example by choosing lipids which are sensitive to temperature. These can be injected systemically into the veins, then placing a heat source near the tumour (although the temperature of the tumour is generally slightly higher than that of neighbouring tissues). 'There is already a real example of this approach', says Piel. 'It is a second generation liposome which is sensitive to heat, using doxorubicin (a chemotherapy agent which has significant cardiac toxicity), which will appear on the market in 2013.' Administration of this product, targeting liver and lung cancer, will be combined with specific techniques to heat the cancerous sites.

It is also possible to activate the active ingredient using light or magnetic stimuli. For example, there is a drug which can be used against retinal macular degeneration, which reacts to illumination of the retina by releasing the active ingredient. Some researchers also dream about incorporating tiny magnetic particles which would react to variations in the magnetic field, or visualising agents (metallic particles) so that medical imaging could check that the active ingredient has arrived at its destination. 'We are encountering many more barriers than we thought at the beginning, which explains why there are not yet many products on the market', summarises Piel. 'We'll need to work together on various levels to be able to effectively deliver active ingredients.' This will also increase their cost ...

Currently, there are only around fifteen specialities which are being marketed in the world, of which 13 are liposome-based. These vectors can only be used in hospital environments.

Truly multidisciplinary

In practical terms, how is a vector produced? 'Chemists create 'tailor-made' polymers', explains Piel. Thus, polymerists from the University of Mons develop for us, new polymers designed to react to a whole series of stimuli. For example, to interact with si-RNAs, which have negative charges, we need polymers which carry positive charges. We therefore combine them with lipids or cationic polymers. The goal is also to find the right relationship to eventually end up with an object which has a slightly positive charge - but not too much, otherwise it is toxic! - because the cell surface contains negative groupings. We therefore need to play on the affinities between the particle and the cell.' In short, a particle has to be produced with the right size, the right charge, and with sufficiently efficient polymers to disguise the active ingredient from the 'eyes' of the opsonines ...

Then, the formulation has to be tested, which is done in close collaboration with other research laboratories. Assessment of the efficiency of the vector is initially conducted on cell culture models before being tested on live subjects, generally mice. Depending on the nature of the project, these stages are conducted either with the Laboratory of Tumor and Development Biology led by Professors Foidart and Noël (LBTD), with the Metastasis Research Laboratory headed up by Professor Castronovo (LRM), or with the Laboratory of Experimental Pathology (Professor Delvenne) (LEP). Clinical trials will take place later ...

Among the projects underway, Piel cites an active targeting project which targets endothelial cells to inhibit tumoral angiogenesis, with a polymeric vector transporting si-RNA, and another using lipidic vectors and si-RNA to inhibit the production of certain proteins in the case of cancers related to the HPV virus, particularly cervical cancers. 'In the latter example, the idea is to exercise prolonged inhibition, so we need to find a system that enables the vector to remain in contact with the cervix for as long as possible. This project is conducted with the support of Télévie; it is still really fundamental, because there is currently no treatment of this kind on the market.' Another project, conducted in collaboration with the Human Genetics Laboratory led by Professor Vincent Bours, aims to administer a natural molecule, which would have interesting anticancerous properties for the treatment of glioblastoma.



That said, the Laboratory of Pharmaceutical Technology  can already boast some encouraging results. 'In partnership with the LBTD, we developed a liposome for pH-sensitive delivery of an active peptide for breast cancer treatment, which allows specific targeting to tumor cells.. The vector operates very well ... but the active molecule has not yet been finalised. So, for the time being, the project is on stand-by.' Another promising project is the 'Carcinom' project which focuses on the administration of genetic material for the treatment of basocellular skin cancers, with activation by phototherapy. 'The project has ended but will be continued through the Télévie project on cervical cancer. The results are encouraging, and we hope one day to end up with something marketable.'

But it doesn't end there!

Don't think for one second that the “galenists” are resting on their laurels! Because, after fine-tuned the ideal vector, they then have to find robust ways of producing it. There is a world of difference between the trial and error potentially leading to success in the lab and industrial manufacture of the same product! 'If one day we want to introduce a drug to the market,' states Piel, 'the manufacturing process must be transposable to the industrial scale, which is no mean feat. Then, the object also needs to be sterilised, bearing in mind that, in general, procedures involving heat cannot be used due to the danger of modifying it. Finally, the vector must also be stable, because it is not feasible to have a drug which has a life expectancy of six months. Liposomes are often unstable in aqueous environments, so they need to be lyophilised, to create a powder which is then dispersed before injection.' The research must be perfect, down to the smallest details ...