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  NANOMEDICINE  -  Treating and healing on three fronts

Despite the very real progress achieved by medicine, researchers and doctors always seem to come up against the same obstacles: illnesses that are diagnosed too late, medicines that are either ineffective or effective but toxic, and the inability to regenerate the organ or tissue damaged by injury or disease. On these three points – and whether in the face of cancers, accident injuries, cardiovascular, neurodegenerative or immune diseases – the ‘nano approach’ promises crucial breakthroughs.

droplets (50nl) dispensing robot. © P. Stroppa, CEA
droplets (50nl) dispensing robot.
© P. Stroppa, CEA
The importance of early diagnosis is well known, especially for cancer. The earlier the disease is identified, the better the chances of combating it with surgery or chemotherapy. But there is a dilemma. While progress in (in vivo) imaging and (in vitro) biochemical and genetic analysis has considerably improved detection possibilities, the examinations are long, costly and sometimes painful for the patient. So, doctors hesitate to prescribe them. It is by rendering these existing diagnostic methods more rapid, reliable, sensitive and economic that this new approach by nanomedicine can help resolve the dilemma.

In vivo viewing

Novel diagnostic approaches: cell surface characterisation of pancreatic tumour cell lines. © J. Schnekenburger, Department of Medicine B,University of Münster

Novel diagnostic approaches: cell surface characterisation of pancreatic tumour cell lines.
© J. Schnekenburger, Department of Medicine B, University of Münster
Real progress has been made in recent years in two fields. The first is in vivo diagnostics. Whether it is scanning, magnetic resonance imaging or tomography, all the techniques for viewing the human body require the injection of tracers or contrast agents.

"Conventional contrast agents make it possible to visualise the anatomy, but are not very effective in evaluating the psychological or molecular processes,” explains Andreas Briel of the Schering Social Research Centre in Germany. “That is why nanotechnologies are so interesting as they make it possible to assemble an inert marker that can be identified by the imaging device and a biological ligand that is able to recognise a particular organ or cell type. Because of their small size, these markers penetrate the tissue more easily and improve the image resolution.” 

On-chip laboratories
The second field is the generalisation of DNA chips. These in vitro devices make it possible to analyse the gene expression of a cell in just a few hours by means of oligonucleides fixed to a solid support that activate a light or electrical signal when they recognise the complementary DNA sequence. Unknown just a decade ago, these chips are now widely used in diagnosing gene expression disturbances in the cells of very small samples which conventional methods were unable to analyse – in the case of biopsies on patients suffering from stomach cancer, for example.   

The ultimate aim of nanomedicine is to identify the tumoural transformation immediately the very first cell is affected. Although there is still a very long way to go, research is beginning to identify the path. Techniques for a rapid analysis of the proteinic combinations of the cell surface – to seek the signature of tumour cells – are at an advanced stage of development. These use protein chips which function on the same principle as DNA chips, but with antibodies that recognise the peptides expressed on the surface of cancerous cells in the place of oligonucleotides.

Gene delivery using ultrasound. The presence of gas in the gene-filled microbubbles allows ultrasound to burst them. © Unger et al. Therapeutic Applications of Microbubbles
Gene delivery using ultrasound. The presence of gas in the gene-filled microbubbles allows ultrasound to burst them.
© Unger et al. Therapeutic Applications of Microbubbles
More futuristically, there is also talk today of ‘on-chip laboratories’. This expression refers to miniaturised systems that make it possible to carry out, in parallel, in the minute cavities fed by microfluids, several hundred biochemical analyses whose results can be studied in real time. “Although considerable sums have been invested in this field, on-chip laboratories have not been developed very much commercially,” says Rutger van Merkerk of Utrecht University, who has just completed a strategic study of the sector’s 20 leading players. “It is a promising technology, but one that it is still at an early stage of development and seeking fields of application.”

The race for miniaturisation, in fact, poses some serious technical problems. Jeremy Lakey, Scientific Director of the UK company Orla Protein Technologies, explains: “To make an interface between the biological systems and electronic devices, the latter must be around the same size as the DNA, membranes and proteins, which means we are talking about scales of under 100 nanometres. This implies we must develop nanofabrication methods in the field of electronics.”

The delivery headache
After the diagnosis comes the treatment. Except in special cases (see box entitled ‘Tumour-killing nanoparticles’), the nano approach is unlikely to result in genuinely new treatments. It does, however, hold the promise of a radical improvement on one key point: the delivery of medicines. For a molecule to be effective – whether the result of conventional chemical syntheses or concocted using advanced biotechnologies – it is not enough for it to be able to improve the condition of the sick organ. It must first reach that organ, which is not easy for large molecules such as proteins.

Gliacells on substrate with electrodes. © Fraunhofer IBMT, St. Ingbert
Gliacells on substrate with electrodes.
© Fraunhofer IBMT, St. Ingbert
“Proteins are being used increasingly as therapeutic agents for several diseases, including cancer, but their development comes up against the problem of passing through biological membranes, their structural fragility and their rapid breakdown in the human body. As a result, today they usually require parenteral administration, which means complicated and painful injections,” stresses Peter Venturini, Director of the Slovenian National Institute of Chemistry.

So does that mean the solution lies in using small molecules that can circulate freely in the body? At one point that may have been the view, but “the slow progress in treating diseases such as cancer using molecules with a low molecular weight brought a change of strategy, with the focus now on administering medicines directly into the affected organs”, explains Costas Kiparissides of the Aristotle University in Thessaloniki (Greece). Gene therapy, on which so many hopes were pinned in the 1980s, also comes up against this problem of delivering the DNA medicines to the target cells.

In addition to this scientific question, there are also economic considerations. As Andreas Jordan, Doctor and Director of the MagForce company, admits, “this research into delivery is also a way of giving a second life to molecules that have entered the public domain, by changing their presentation”. 

Transport engineering
So how can the nano approach revolutionise the delivery of medicines? The answer lies in a very simple geometric fact: for a given mass, the more a substance is contained in small particles, the greater the total surface area with a biological activity able to interact with the receptors on the cell surface.

This explains the attention focused on reducing the size of the systems envisaged for transporting the medicine to its target organ – for example, in tiny bubbles encapsulating the therapeutic molecule made up of single-layer lipids (micelles) or multi-layer lipids (liposomes), or otherwise coated in biodegradable polymers packed with antibodies able to recognise the target cell. All of which brings us to the realms of the very fine engineering of molecular ‘transport’ vehicles that must, at the same time, protect the medicines from breakdown. 

As Richard Aljones, of Sheffield University, likes to say, “the most wonderful example of nanotechnology that we know is none other than the living cell, that constructs itself all alone by a process of self-assembly of its parts”. To build these devices, an initial approach therefore is to draw inspiration from the principles at work in living nanometric systems, such as ribosomes or membranous enzymes. Ultimately, it is even envisaged to equip these nanoparticles with remote delivery instructions, so as to trigger the medicine release (by means of electromagnetic waves or infrared stimulation, for example) once the vehicles reach their targets.

The miniaturisation that permits this nano approach also has another advantage in that it makes it possible to envisage innovative ways of administering medicines that are more practical, effective and/or less painful, such as pulmonary administration with nanoparticle sprays or transdermal administration, in particular for unconscious patients. 

Left: Intervertebral disc, 12 months after treatment with autologous disc chondrocytes.Right: untreated intervertebral disc.Regenerated discs mimic native disc morphology; autologous treatment promotes tissue regeneration. © T. Ganey, co.don AG
Left: Intervertebral disc, 12 months after treatment with autologous disc chondrocytes.Right: untreated intervertebral disc.Regenerated discs mimic native disc morphology; autologous treatment promotes tissue regeneration.
© T. Ganey, co.don AG
The third and final field of application for nanomedicine is regenerative medicine which aims to help the body heal itself. The first stage was the replacement of defective organs, which appeared in the 1970s when the first materials for implantation in the human body were developed. But these were no more than inert ‘spare parts’ that were not biodegradable and had also often been developed for quite different applications. In the mid-1980s, a second generation of ceramic- and glass-based materials became available. These were either biodegradable (once the lesion repaired) or able to stimulate autoregeneration action – but never both at the same time. The challenge for today’s researchers is to combine these two properties of biodegradability and bioactivity in a single structure. When working on a nanometric scale it is indeed possible to design combinations of inert bodies and biological molecules previously inaccessible to conventional chemistry.

Cellular differentiation, intelligent materials
Another avenue is also opening up for a command of cellular differentiation, at the heart of stem cell research. “In the traditional approach to cell therapy, the cells are grown in a liquid environment, which limits their differentiation possibilities. We are currently seeking to grow them on a solid surface covered on the nanometric scale with combinations of proteins able to induce their transformation into a desired cell type,” explains Günter Fuhr of the Fraunhofer Institute for Biological Engineering (St Ingbert, DE) and coordinator of the European project CellPROM (Cell Programming by Nanoscaled Devices).

Finally, as we saw in connection with medicine delivery, the nano approach can permit the design of ‘intelligent’ materials able to adapt their behaviour to local biological conditions or external stimulations. “Such materials, when used as both a nutritive and structural matrix, could serve to multiply healthy cells so as to then reimplant them in the diseased organ,” comments Alessandra Pavesio, of the Italian company Fidia Advanced Polymers, who is working on the application of this principle for meniscus regeneration in the European Meniscus project. 

Like many industrialists in this sector, Pavesio wants the European regulations to be made more flexible so as to speed up clinical trials on nanomedicine products. David Rickerby, of the European Commission’s Joint Research Centre Institute at Ispra (IT), nevertheless believes that “the present legal framework, which does not differentiate between nanomedicine and conventional products, is sufficiently flexible to integrate innovations of this type at their present stage of development, even if regular evaluation will have to be made to allow for scientific progress”. So it seems that the law will not be caught off guard by the expected scientific and technical changes brought by nanomedicine.

  Towards artificial corneas?  
  The cornea that protects the surface of the eye consists of two layers of epithelial cells surrounding a proteinic and cellular matrix (the stroma) of complex structure that is both transparent and very rigid. Corneal lesions are responsible for about 6 million cases of blindness worldwide. Every year in Europe over 27 000 cornea grafts are carried out, but the lack of donors, problems of immunological compatibility, and risk of disease transmission all mean that this method does not give full satisfaction. Therefore, the ultimate aim of the 14 teams from nine European countries working within the Cornea Engineering consortium is to construct artificial corneas for these ophthalmologic grafts. 

Artificial corneas could also serve to test the ophthalmologic toxicity of cosmetics and thus reduce the need for experiments on animals. “The Italian consortium team member has already succeeded in reconstructing the epithelial layer by means of autologous cell grafts. The next step is to reconstruct the stroma. By assembling protein fibres, on the nanometric scale, the aim is to develop a matrix within which the stem cells can be differentiated,” explains David Hulmes, of the Institut de Biologie et de Chimie des Protéines in Lyons (FR), the Project Coordinator.


  Tumour-killing nanoparticles  
  Destroying tumours with heat is not a new idea, but for years it has faced the major obstacle of side effects due to the difficulty of restricting the hyperthermia to the tumour region alone. The Berlin company Magforce, founded in 1997, has now come up with an original solution to the problem. It is based on the injection of iron oxide nanoparticles with a special protein coating that ensures they are only absorbed by the tumour cells which are then unable to excrete them. “Once they penetrate these cells, the nanoparticles act as tiny receiver aerials,” explains Andreas Jordan, the company’s founder. “The application of a magnetic field then creates a local hyperthermia that kills the tumour cells.”

Successful phase 1 human clinical trials have already been carried out using this promising technique. Phase 2 trials, aimed at demonstrating effectiveness in certain cases of prostrate and brain cancer, are currently being carried out at the Charity Hospital in Berlin. In the light of the very encouraging results, MagForce expects this new treatment to be marketed by 2007. Work is already in progress on a new generation of these nanoparticles. When incorporated in proteins that specifically target tumour cells, these can be injected directly into the bloodstream rather than into the tumour, as is the practice today.