Correlating the macroscopic properties of a massive material with the infinitesimal dimensions of its individual components is not an immediate or intuitive task. Still, the relationship between what is macroscopic and what is microscopic is far from being a weak one. Indeed, it is well known that the proprieties of materials depend on the nature and on the reciprocal arrangement of their atoms or of their individual constituents.
Even in the most painstaking job conducted on a massive material, millions of atoms are manipulated and set in motion. Furthermore, theoretical analyses show that it is possible, without breaking the fundamental rules of physics, to rearrange the basic constituents, one by one, in a similar way to that employed with kids’ bricks, to create new systems.
This type of approach, more frequently referred to as nanotechnology or molecular manufacturing, when adequately optimised, allows the construction of stronger, cheaper, lighter and “smarter” devices.
Over the last twenty years, the trend towards miniaturisation has made it possible to hurdle the “micro”-related difficulties and to enter the “nano” universe. This is a technological challenge stimulated by a growing request for increasingly smaller machines and considerably supported by substantial economical investments by all industrialised countries. Suffice it to mention that during the Fifth Framework Programme of the European Union only, the financial support offered for activities related to nanotechnology settled on approximately 15 billion Euro.
If we take into account its incredibly vast and manifold application potentials, we can reasonably predict that over the next decades nanotechnology will become a major strategic research guideline in all industrialised countries. Designing new planning and synthetic strategies therefore becomes a must in order to allow the approach to versatile production methods for the development of materials and devices displaying the desired proprieties and functions.
All the synthetic methods employed for the realisation of nanosystems may be classified according to two general and antithetic approaches. In the top-down approach, by starting from massive objects and gradually reducing them through processes, such as for instance mechanical crushing or photolithography, you can reach dimensions in the range of dozens of nanometres.
On the other hand, the bottom-up approach starts from the bottom, and is based on the manipulation of atoms and molecules which, when adequately assembled, will result in those molecular building blocks which represent the basic constituents of the final structure. In a stage which, in scientific terms, is still at a pioneering level, but at the same time is proving really feverish in terms of commitment and resources made available, the challenge we are now faced with is that of exploiting the tools made available by nanotechnology in order to manipulate the biomolecules regulating life and death, as well as health and diseases.
The keystone for the materialisation of such efforts is to acquire the fact-finding and technological tools required to modulate devices and materials to the manometer scale and to design machines and tools that are not larger than biomolecules, such as for instance DNA.
Micrometric (and in a not too distant future nanometric) sized devices disclose incredibly vast potentials within the field of biomedicine, a field in which biology, engineering and medicine converge into a territory which is stilly chiefly unexplored, but fascinating. The common goal is that of designing systems that may provide therapeutic effectiveness in treating a great variety of pathologies, ranging from tumours to heart diseases and diabetes. The new doors that are opening at the moment are also the result for progress chiefly attained in the knowledge of the basic mechanisms: indeed, it has been the understanding of biological processes at a molecular level that has allowed an engineering approach to complex natural systems into which, as already mentioned, a variety of disciplines converge.
There still is some ambiguity as far as terminology is concerned, leading novices to mistake bioengineering with biomedical engineering: whereas the latter represents an application field, even though distinctively involving a variety of disciplines, the former is in fact a new but rapidly expanding discipline. In this connection, the ability to modulate the structure of devices to the nanometre scale will make it possible to revolutionise medicine and related biomedical disciplines.
The reason for this is a simple but not trivial one: therapeutic effectiveness always requires, to a certain degree, the imitation of the biological structures involved in that specific process, and the size of such structures is very close to the nanometre scale. In this field, a key role is currently played by photolithography.
With the aid of photolithography, it has been possible to place single strands of DNA on a chip in known and specific positions. The probes react in a specific manner with individual DNA elements marked by a fluorescent substance, and these are thereby detected. In this way, by keeping track of the areas of the chip in which the fluorescence is visible, you can trace back the biochemical identity of the sample DNA.
Therefore photolithography, which is a basic technology in microelectronics and in MEMS (MicroElectronic Mechanical Systems), proves to be an extremely brilliant and innovatory approach for a high-speed analysis of nucleic acids. And it also discloses interesting practical and practicable applications, such as the high-throughput production of new drugs, the evaluation of genetic risks, legal medicine analyses, identification of individuals or preventive monitoring of germ warfare. And we should not forget that this type of technology has allowed the materialisation of the “chip laboratory” concept, that is the miniaturisation of testing and diagnostic tools (sensors) to a point whereby they can now be functionally included and integrated within portable microdevices. These types of technologies, based on photolithography, are called biomedical microtechnologies or bioMEMS.
However, the reader should not be misled by the “micro” prefix: over the last few years, the photolithography resolution limit has been brought up to hundreds of nanometres, micro-electronic-related processes within the “nano” universe. And this has taken place despite the fact that photolithography is traditionally numbered among top-down processes, since it originates from macroscopic objects, such as for instance layers of material depositing on silicon wafers, and it removes in a gradual and controlled manner specifically defined sections, thus creating on the substrate objects with lateral sizes that are reduced to 200 nm and a few angstroms in thickness. As a matter of fact, biomedical research has not yet got involved in the nanometre perspective. In order to attain this goal, researchers are acquiring methods and skills relating to other fields, such as the semiconductor technology, a sector in which the production of chips having a size of a few hundred nanometres had been for several years a consolidated routine. It was in our laboratories of the Ohio State University that we acquired these manufacturing techniques in order to manufacture devices that may be effectively applied in the treatment of diseases such as, for instance, diabetes mellitus, which is characterised by an insufficient secretion of insulin by the pancreas. As we shall more extensively explain further on, we have analysed silicon microcapsules, capable of bringing about the replacement of diseased cells by healthy cells.
For instance, in the above-mentioned case of diabetes, when the cells are not working properly, capsules containing healthy replacement cells may be implanted under the patient’s skin. Supplying the body with new cells represents a very effective method for the treatment of certain diseases, such as those caused by an enzyme or hormone deficiency.
The research work we have embarked on over the last few years in our university laboratories and in the laboratories of iMEDD (“intelligent MicroEngineered Drug Delivery”), a spin off company established to make available on the market the products of technology within this sector, has chiefly focused on the employment of micro- and nanotechnology for the realisation of multifunctional devices for therapeutic purposes.
An example is provided by intelligent devices for drug release, which, if suitably implanted, are able to localise in the area affected by the disease, and to release the substance in a manner that is consistent with external stimuli and biological signals. In this connection, the testing work conducted is chiefly related to the micro-manufacturing of devices that can be broken down into two different classes:
1) membranes with nano-pores;
2) multifunctional organelles (particles) for the release of therapeutic agents. As regards the first range of systems, over the last seven years in our laboratories numerous variants of such silicon membranes have been manufactured, which have in common the employment of a spacing (or rather a sacrificial) silica layer placed between two structural silicon layers.
Once the sacrificial layer has carried out its “moulding” function, it can be easily removed by means of selective chemical etching, which has no impact on the silicon layers. In this way, the pore pattern faithfully and accurately reproduces the pattern of the sacrificial layer, whose microstructure can in turn be controlled with nanometric accuracy. This type of technology, based on the resort to a sacrificial layer, draws inspiration from the “grid oxides” broadly employed in the microprocessor technology. And, within a much broader time scale, it refers to the techniques based on the employment of wax moulds by ancient Greek artists in forging their sculptures. These nano-pores have immediately proved effective in molecular-scale separation, such as in the case of virus extraction from biological fluids. On the other hand, our chief application is based on the use of cellular bio-reactors, which can be implanted for a physiologically–controlled drug release. The nano-membranes can also be employed as agents capable of regulating the release, over extend time intervals, of biopharmaceutical products from the injected depots. This last application takes place through a passive molecule mass transport along channel provided with a diameter which is only slightly greater than that of the molecules themselves. The processes is based on diffusion, but it also involves a whole range of much more complex and interdependent phenomena, such as for instance the interaction of the molecules with the pore walls, a phenomenon which is referred to as “single-file diffusion”. Besides, the attainment of an active and convective-type mass transport, which would be desirable in a distance or self-regulated controlled dispenser, is in fact a goal which is made practically impossible by the laws of physics and by the features of the systems involved: the pressure required to push a fluid through channels having a nanometre-scale diameter is superior by various orders of magnitude to the actual maximum stress that the materials and structures in question can bear. Such considerations show how mechanical engineering may play a leading role in the design and realisation of highly innovative biomedical nanotechnologies. This approach is mediated by nanotechnology, which would also appear to prove highly effective in genic therapy, where fully reliable methods are required to supply genes to human cells. In theory, many devastating and invasive diseases could be cured by introducing DNA fragments to “repair” missing or defective genes. In practice, however, the introduction of new genetic material inside a cell brings about strong and often lethal immune reactions. Indeed, the immune response represents the greatest bar to the application of the genic therapy. The replacement cells are alien to the body, and are therefore immediately recognised and attacked by the immune system of the host body, with often devastating consequences. The tools made available by nanotechnology again make it possible to get round the difficulty. The possibility of blocking antibodies by means of an artificial barrier would prevent the immune system from recognising the transplanted cells. This is why we have again resorted to silicon capsules incorporating membranes with pores that are small enough not to let antibodies in, but sufficiently large to allow the inflow and outflow of the desired cells. The idea is as brilliant as it is difficult to carry out: attaining nano-pores that are small enough to prevent the access of antibodies involves reaching a size of just a few nanometres (apparently the size of an antibody is of approximately 18 nanometres). Current photolithography makes it possible to produce circuits in an order of magnitude of a few hundred nanometres. By adjusting such methods, however, we have succeeded in creating pores with a diameter of just a few nanometres.


Fig.1 A submicroscopic drug delivery device uses a membrane with pores as small as 6 nm to protect therapeutic substances from attack by the body’s immune system.

As already mentioned, once this method will have been optimised, the opportunity will disclose to apply it to one of the most widespread pathologies, that is diabetes. In one of the forms of this disease, the pancreatic cells producing insulin do not work properly.
One of the most effective therapies would involve implanting, directly into the body, copies of the insulin “factories”, the so-called “Langerhans Islands”. These would replace the defective pancreatic functions and reactivate the normal insulin-production process. The implantation of cells from different organisms, usually swine, will however bring about an immune reaction, thus making it necessary to administer, at the same time, immune suppressors; these however put the patient in a vulnerable situation and expose him/her to infections. In order to avoid this drawback, our strategy involves the resort to release devices manufactured with nano-porous membranes. In this way, the small glucose molecules may freely spread through the nano-pores of the capsule and activate the cell, thus restoring control over insulin production. This approach is already proving successful on small animals, but it will need to be further optimised and adapted in order to also work on larger animals, such as for instance dogs, before being tested on human beings.
Nanotechnology offers versatile and effective tools also for the treatment of tumours, an area in which rapid and significant progress is required. The range and degree of the devastating and invasive side effects of chemotherapy are tragically well known. The chief contraindication associated with the use of conventional chemotherapeutic drugs is related to the fact that only a relatively small amount of the administered substances reaches the tumorous cells, whereas the greatest amount attacks hair follicles, the immune system and the tissues, thus bringing about the well known side effects: hair loss, nausea and weakness.



Fig.2 With lateral dimensions of one micron or less, the depicted particle is smaller than any blood cell. Once a concentration of these particles is safely injected in the bloodstream, they travel freely through the circulatory system. In order to direct these drug-delivery microparticles to cancer sites, their external surfaces are chemically modified to carry molecules that have lock-and-key binding specificity with molecules that are expressed preferentially on the blood vessels that support a growing cancer mass. Here is how these microparticles may provide a revolutionary new approach to the fight against cancer: As soon as the particles “dock” on the cells that line the internal surface of these blood vessels, a compound is released that forms a pore on the membrane of these cells. This leads to cell death, the consequent collapse of the entire blood vessel, and ultimately to the death of the cancer mass that was nourished by the blood vessel. In view of the targeting specificity of the molecules on the surface of the microdevice, very little or no collateral damage is done to healthy tissue, and thus the treatment may be repeated multiple times, as needed.

Such effects have made it necessary to identify and test a more effective and less invasive therapeutic approach. Indeed, the versatility displayed by the modern micro- and nanotechnologies have made it possible to create the devices which have been picturesquely but effectively described as “chip pharmacies”.
These are pills incorporating different functions, which are however interconnected and interacting, capable of releasing in a controlled manner the desired substance into the affected organ. As a small but skilful Trojan horse, when taken by mouth, this pill goes through the body undisturbed and unnoticed and settles into the affected organ, where it subsequently releases in a controlled manner the anti-tumorous drug which will destroy the carcinogenic cells without bringing about further or undesired effects. This challenge, which is as daring as it is stimulating, does not simply consist in producing drug release in the right place, but also at the right time.
Our research work is focussing on time controlled delivery, that is on a controlled release of the substance, which may allow for more complex release schedules that are not only based on the “space” variable but also on the other therapeutically crucial factor: that is “time”. The secret lies in dosing the drug in such a way that the amount released localises exactly inside the so-called “therapeutic window”, under which no perceptible effect is gained, and above which overdose occurs, with its counterproductive and often detrimental effects. In addition to the above-mentioned functional conditions (controlled drug delivery both in space and time), these devices are also to meet certain basic requirements, which will ensure their therapeutic use is actually practicable: they are to withstand the drastic pH changes occurring in certain organs (as in the case of the strongly acid environment existing in the stomach); in addition, they are to offer high mechanic resistance and biocompatibility. Again within the field associated with fighting tumours, another project started by our laboratories relates to the creation of an intravascular microparticle with a side dimension smaller than one micron.

Fig.3 OSU’s world-leading heart surgeons, Prof.s Michler and Wolf, demonstrate that the biochip may be implanted in the heart by a computerized robot. This powerful combination of robotic surgery and nanotechnology is directed at revascularizing infarcted heart tissue - that is, restoring heart function following a heart attack. There is only one place in the world that is equipped to carry out this revolutionary vision: Ohio State University.

When injected in an adequate concentration into the bloodstream, these microcapsules may move through the circulatory system and finally reach their target, that is the carcinogenic cells. In order to make such microparticles selective, their surface is chemically functionalised by means of molecules possessing a lock and key binding specificity for the molecules coming from the blood vessels nourishing the tumorous mass. It is indeed this selectivity that provides a revolutionary approach to tumour therapy.
Once the particles hook into the cells delimitating the internal surface of such vessels, they release a compound which produces a hole in the membrane of such cells.
This brings about the death of the cell and the consequent collapse of the entire blood vessel, which in turn results in the death of the tumorous mass nourished by the blood vessel in question. Thanks to the specificity of the molecules on the surface of this microdevice, the damage caused to the healthy tissue is extremely limited and treatment can be repeated time after time, if required.
This practice would consent patients to relieve many pains due to traditional tratments. Nanothecnologies serve this purpose, too.

Mauro Ferrari Ph.D.
Director of the Biomedical Engineering Center
at The Ohio State University-US

Silvia Gross
Ricercatore at Institute of Molecular Sciences and Technologies (ISTM) of the Italian National Research Council (CNR)