About the development of nanobiotechnology

The article was published in the journal "Innovations" No. 12-2007

About the development of nanobiotechnologyM.P.Kirpichnikov, K.V.Shaitan

Lomonosov Moscow State University

Faculty of Biology, Moscow 119991, Leninskie gory, 1, p.12

The article summarizes some of the current trends in modern nanobiotechnology. Attention is paid to the issues of targeted drug delivery, disease diagnostics, biocompatible materials and materials based on web proteins, nanodevices and nanorobots, self-assembly of nanobiostructures, molecular modeling and computer design of nano- and biostructures, potential biological risks arising from the use of nanoparticles and nanomaterials, problems of personnel training for the nanobioindustry and bioengineering.

Currently, considerable attention is being paid to the development of nanotechnology all over the world, organizational structures are being formed and priorities are being chosen. Nanotechnology presents a wide range of scientific achievements, with outlets in a variety of industries and fields of activity. Typing, for example, the word "nano" on Google's website, we will get about 70 million links.

The number of references to the word "nanobio" is about 176 thousand. Therefore, in this article we will only briefly touch on some issues of nanobiotechnology, i.e. the nanotechnology section, which is related to biology and medicine. Below is a very brief overview of certain areas of nanobiotechnology, in which, in our opinion, there is already a significant reserve in Russia and where we can expect the creation of competitive technologies and products. Taking into account the general informational nature of the article, we basically refused to refer to any special scientific works.

Additional general information can be found, for example, on the Internet using search engines.

Figure 1 shows a far from complete range of directions for the development of nanobiotechnology, which, as we believe, are very promising. 

Fig.1. Some promising directions of nanobiotechnology development.

 1. Targeted drug delivery

One of the areas of intensive development of nanobiotechnology applied to biomedicine is the development of new methods of selective intracellular and intracellular delivery of physiologically active substances. The basic idea of targeted delivery is shown in Fig. 2.

Fig.2. Nanoconstructions for selective intracellular drug delivery.

There are several directions here. A number of nanoscale forms of carbon (fullerenes, nanotubes) have a good penetrating ability with respect to biomembranes and, most importantly, are able to overcome the blood-brain barrier and act as transporters for drugs.

Preliminary studies of transmembrane transport of nanostructures and their complexes with biopolymers conducted, in particular, at Lomonosov Moscow State University show the fundamental possibility of creating nanodevices (nanomachines) for selective recognition of receptor sites on the cell surface and delivery of active substances into them, which in the future can expand the therapeutic possibilities of treating oncological diseases, neurodegenerative diseases, neuroinfections et al . and dramatically reduce the therapeutic doses of drugs, minimizing side effects.

An important direction is associated with micro- and nanocapsulation of biologically active substances (BAS) based on biodegradable polymers, as well as with the use of liposomes. The development of this technology is carried out by a number of institutes of the Russian Academy of Sciences, the Russian Academy of Medical Sciences and Moscow State University, which will allow in the future to create fundamentally new drugs with controlled therapeutic effects on certain tissues and organs. This technology is used to create effective drug systems for the controlled excretion of encapsulated BAS: drugs (including insoluble in water or unstable), peptides and proteins (having the functions of hormones and cytokines), as well as genetic constructs carrying genes of enzymes, hormones and cytokines.

Progress in the development of the synthesis of biodegradable polymers, nanocapsulation technology, as well as methods of cellular and molecular physiology and pathophysiology will allow the use of technology for the creation of systems for the controlled excretion of BAS based on nanoparticles from biodegradable polymers for the treatment of cancer, hormonal disorders of various etymologies, atherosclerosis, diabetes, tuberculosis and other socially significant diseases, as well as for gene therapy of a wide range of spectrum of diseases.

The technology of including drugs in nanocapsules will allow the use of many medicinal compounds, the delivery of which to organs and tissues would be very difficult due to their insolubility in water or instability; this technology will reduce toxicity and achieve the desired pharmacokinetics for drugs. Nanocapsulation of proteins and nucleic acids will make it possible to create systems for the delivery of peptide hormones, cytokines and genetic constructs to various tissues of the body. The development of ways to attach targeted ligands to nanoparticles will help deliver biologically active substances to certain tissues.

An important point is also the study of the transport of nanoobjects of metallic and semiconductor nature, as well as superparamagnetic nanoparticles for selective destruction of cells during electromagnetic heating, which is important for the treatment of a number of tumors.

2. Nanodiagnostics of pathological conditions and infections, nanobiosensorsThe successes of recent years in describing the functioning of the human genome, the molecular mechanisms of cellular processes provide the basis for a significant increase in the informativeness of medical diagnostics.

Instead of controlling a few compounds, traditionally interpreted as characteristic markers for a particular disease, it becomes possible to obtain reliable and informative information about the functioning of the body and the development of the pathological process based on a comprehensive account of the level of a significant number of compounds in one way or another associated with the pathological process.

In this regard, it is extremely important to reduce the complexity of obtaining diagnostically significant information, thereby ensuring the possibility of mass application of new methods. The necessary measures to ensure the effectiveness of the analysis should include reducing the volume of samples, the use of unified protocols for analysis, reducing its duration and automated registration of results.

To date, among the developments in this direction, the creation of microchips dominates, in which a fluorescent label, chemically conjugated with specific reagents, binds to certain areas of the carrier substrate. The analysis capabilities are significantly expanded when colloidal nanoparticles are used as detectable markers.

Varying the composition and sizes of nanoparticles of gold, silver and other metals makes it possible to form a number of markers with different absorption spectra. An even greater variety of markers is provided by using so–called quantum dots - fluorescent nanoparticles based on cadmium, zinc and other metals. Quantum dots with different particle diameters (achieved by modifying the synthesis conditions) cover the entire visible spectrum of radiation, creating an effective basis for multianalysis.

Thus, the use of nanoparticles for labeling specific complexes of the compound being determined (for example, by immobilizing specific antibodies on their surface) allows simultaneously quantifying the content of various compounds in the test sample. The principal feature of this approach is the ability to determine several compounds in one sample. This makes it possible not only to reduce the sample volume, but also to carry out differential detection of structurally similar compounds capable of binding to the same receptor elements.

Atomic force microscopy has significant diagnostic capabilities (Fig.3,4), which is used to detect complexes of biomacromolecules, for example, protein-antibody.

Fig. 3. Detection of AFM antigen-antibody complexes

Fig.4. Images of antibodies in the free state and immune complexes obtained using an atomic force microscope.

To create highly sensitive biosensors, a technology using biopolymer functional films in a hydrogel state is also promising. The surface architecture formed by them has great advantages for use as biosensors due to sensitivity to changes in external environmental parameters, biocompatibility, and the possibility of selective DNA capture.

3. Nanostructured biocompatible materials

Available experimental and clinical studies show that plasmon-coated coatings on the surface of implants stimulate osseointegration well and are a very effective solution to the problem of implant rejection. However, coatings on implants should have a complex of often mutually exclusive properties: high adhesion, porosity, developed morphology, good biocompatibility.

These conditions are achieved by using layered nanostructured coatings. It is known that the formation of the specified properties of materials is possible by creating conditions for the formation of self-organizing structures of the nano-range. However, in relation to the processes of plasma deposition of bio-coatings on implants, the conditions for the formation of nanostructures have been little studied. Meanwhile, the transition to a new level of interaction between artificial (coated implant) and natural (bone tissue) materials would qualitatively improve the process of osseointegration of implants and increase the biological contact of the implant and the bone bed.

Therefore, it is important to improve methods and technologies for obtaining bio-coatings, as well as to find new ways to form the specified properties of materials. It is also necessary to improve research complexes in order to obtain a reliable knowledge base about such complex multifactorial processes by studying the influence of nanostructured formations on the mechanical and physico-chemical properties of the coatings formed.

Of great interest are biocompatible materials based on the spider web proteins (spidroin 1 and spidroin 2), which form fibers consisting of nanofibrils in the spider glands. These molecular structures, selected by Nature in the process of evolution, are able to suggest us successful solutions to many technical problems. Spider fiber has unique properties and has a huge innovative potential in a wide variety of industries.

Viscoelastic spider silk frame threads have both high tensile strength, exceeding steel and comparable to Kevlar, and high elasticity. This leads to very high values of the rupture energy, for which this material has no analogues among other natural and artificial materials.

The web proteins have a very interesting structure. There is an alternation of hydrophilic and hydrophobic segments, which is extremely important for ensuring solubility and regulating the aggregation of molecules, which leads to the formation of nanofibrils, namely, the presence of nanofibrils ensures the presence of unique mechanical properties of this thread.

Molecular modeling of the structure of nanofibrils carried out at the Department of Bioengineering of the Faculty of Biology of Moscow State University showed that the high energy of rupture is apparently due to the formation of supercoils from beta layers rich in polyalanine inserts of speedroins (Fig. 5).
Fig.5. Formation of a superspiral from beta layers of polyalanine fragments of speedroins.

There are first results that indicate the fundamental possibility of obtaining artificial filaments and films based on recombinant analogues of spider web proteins, comparable in their properties to natural filaments, as well as the possibility of developing a technology for creating biocompatible materials with unique properties.

4. Molecular machines, self-assembly of nano- and nanobiostructures, molecular modeling and design of functional nanostructures and their complexes with biopolymersNow they are increasingly talking about the nanomir, about the laws of the nanomir, about its practical use.

I would like to emphasize the great generality of the nanomir. In particular, the main "bricks" from which living organisms are built are the brightest representatives of this nanoworld. Enzymes, receptors, ion channels, DNA, RNA, ribosomes, molecular motors and many other components of the cell and the organism as a whole are nanoparticles in terms of their size, but at the same time extremely "smart" and from a functional point of view very rationally made nanoobjects.

A few words about the possibilities of nanobiotechnology for molecular electronics. Bioelectronics or bio-optoelectronics as a branch of molecular electronics tries to apply biostructures, primarily proteins or their modified analogues, as light-controlled modules of computer and optical devices. The main requirement for biological modules (chips) is that in response to physical exposure — light, they must reversibly change their conformation and form at least two discrete states. These states should differ in easily measurable parameters, such as absorption spectra.

The most likely candidates for the role of such chips are photosensitive membrane proteins that initiate and ensure the operation of the most complex molecular "machinery" of two basic photobiological processes – vision and photosynthesis. Specifically, we are talking about the molecules of the visual pigment rhodopsin and bacteriorhodopsin – a protein from halophilic microorganisms.

Both of these proteins have photochromism and can be considered as the basis for the creation of photochromic materials (chips) of bio-optoelectronics.

Bacteriorhodopsin, unlike other biological structures, has sufficiently high thermal and chemical stability to become the first, but by no means the last, photosensitive material of biological origin suitable for industrial use in optoelectronic devices.

An impressive example of this is the recent announcement about the development of a fundamentally new media that allows you to record up to 50 terabytes of information on a conventional laser disc, that is, about ten thousand times more than on a traditional DVD. This new information carrier is bacteriorhodopsin.

As a cell storing one bit of information, two states of bacteriorhodopsin are used, which can pass one into the other under the action of light of a certain wavelength. At the same time, we are not talking about the usual (wild type), but a genetically modified bacteriorhodopsin with exceptionally high thermal and chemical stability. Both states of the bacteriorhodopsin molecule can persist indefinitely – at least several years. This makes it possible to record information using a laser on protein molecules in the form of a two-dimensional or linear sequence of zeros (the initial state of bacteriorhodopsin) and ones (the product of its phototransformation), and then save and read it.

The principles of the organization of biological systems are very effective and evolutionarily selected for the creation of nanometer-sized devices with a given function. Objectively, the use of these principles in nanotechnology is at the stage of exploratory development.

At the same time, it is already clear that designing and creating molecular machines that use the energy of chemical reactions (or changes in the composition of the medium) to perform elementary functions – photo switches, moving a molecular object, creating a potential difference, developing traction is a real task. This area is, in particular, a priority for the European program for the development of nanotechnology.

Considerable experience has been accumulated in Russia in the detailed study of molecular mechanisms of elementary biochemical and biophysical processes (ligand diffusion, electron transfer, proton transfer, conformational rearrangements during chemical transformations, enzyme-substrate interactions, ion channel operation, etc.), the dynamic theory of electron-conformational interactions as the basis for the functioning of molecular machines has been developed, the fundamentals of molecular modeling and design of nanodevices.

Works on the physical theory of self-organization of protein structures have been widely recognized. This makes it very likely that breakthrough work will be carried out to create molecular machines and functional nanostructures and gain competitive advantages in this area.

In connection with the problem of creating nanodevices, nanorobots, etc.  It is impossible not to dwell on the issues of molecular modeling and molecular design of nano- and nanobiostructures. The rapid development of computer technology, the near-term availability of supercomputers and the accumulation of reliable data on interatomic and intermolecular interactions makes computational experiments with nano- and biostructures a source of objective information about their physicochemical properties.

Moreover, the level of detail of processes involving nanostructures obtained in numerical experiments is as detailed as possible. The relatively low cost of computational experiments compared to their real counterparts makes molecular modeling methods the main tool for designing nano- and nanobiodevices and structures.

Figure 6 shows an example of self-assembly of a nano-sample consisting of a carbon nanotube and polyalanine. This system was proposed for targeted delivery of BAS and calculated by molecular dynamics methods at the Department of Bioengineering of the Faculty of Biology of Moscow State University. From a fundamental point of view, this system is interesting as, in fact, the first example of self-assembly of a functional nanobiodevice (nanorobot). The polypeptide molecule adsorbed on the outer surface of the nanotube (closed at one end) due to thermal fluctuations is spontaneously packed inside the nanotube in a conformation close to the alpha helix.

That is, there is a charging of the nano-matrix. If a molecule (molecules) was previously packed into a nanotube, which disintegrate under external influence or dramatically increase the occupied volume, then a kind of "shot" occurs and the system works like a nanopun. Figure 7 shows the result of a numerical experiment on the effect of a nanowell on a biological membrane: a polypeptide molecule penetrates into the biomembrane in a short time. By covering the mouth of the nanotube pore with ligands specific to certain receptors on the cell surface, it is possible, in principle, to achieve selectivity in the delivery of BAS (in this case, a polypeptide).

5. Potential risks from the use of nanoparticles and nanomaterialsWe are surrounded by a huge number of nanoparticles that are the product of ordinary human activity and most of them are apparently relatively harmless.

At the same time, it is now reliably established that in the nanoscale state, many quite harmless substances become biologically very active and, in many cases, highly toxic.

Examples of asbestos, uranium oxides, fullerenes and many other nanoparticles are well known. However, there are still no systematic studies on the nature of the toxicity (and carcinogenicity) of nanoparticles, there are no certified technologies for determining this type of toxicity, there are no (with rare exceptions) appropriate sanitary and hygienic standards for the use of nanomaterials.

This was recently brought to the attention of the Ministry of Consumer Protection (letter dated 02.06.2007 No. 0100/4502-07-32 ).

We note the main factors causing potential risks from the use of nanoparticles and nanomaterialssmall size and ability to penetrate barriers (including blood-brain barrier)

• large specific surface area
• abnormal reactivity (generation of free radicals)
• facilitating the penetration of molecules of other substances
• features of metabolism (macrophages "do not see" sizes < 70nm)
• constancy to the accumulation of a number of nanoparticles.

The issues of biological (as well as environmental) risks in the use of nanomaterials are important in predicting the effectiveness of the introduction of nanotechnology. It is also necessary to take into account the possible impact of nanoparticles and nanomaterials on the overall state of affairs with ensuring biological and chemical safety as one of the most important areas of strengthening the national security of the Russian Federation, set out in the "Fundamentals of State policy in the field of chemical and biological safety of the Russian Federation for the period up to 2010 and beyond" (approved by the President of the Russian Federation 4 December 2003 No. PR – 2194). Chemical and biological safety is of particular importance in modern conditions due to the intensification of terrorist manifestations, which can be aimed at selectively affecting biological systems and organisms. 

The analysis of the current state shows that, on the one hand, new "breakthrough" effective technologies and materials are being created on the basis of scientific developments in the field of nanotechnology. On the other hand, the development of nanotechnology may lead to the creation of a new class of chemical and biological weapons using the properties of nanoparticles. Currently, there are no systematic methods for detecting nanoparticles in the environment and biological objects.

Therefore, the development of technologies for determining the risks from the use of nanoparticles and nanomaterials, their certification on this basis is of crucial importance not only for the development of new industries, but also from the point of view of ensuring national security. It should also be borne in mind that in the last few years, close attention has been paid to the issues of nanorisks around the world, national programs, non-governmental and international organizations on this issue are being created, a special journal Nanorisk is being published.

It should be noted that the issues of emerging risks from the use of specific nanoparticles should be seriously and methodically investigated in special laboratories, and unprofessional generalizations in this matter have no basis.

Currently, based on the developments of a number of institutes of the Russian Academy of Sciences, RAMS, MSU, etc., there are all prerequisites for the rapid and effective development of technology for determining potential risks from the use of nanoparticles and nanomaterials.

5. Development of the educational component of nanobiotechnologyThe development of nanobiotechnology, integrating knowledge and skills from many disciplines in a new combination, requires certain measures to train specialists.

First of all, it is a focus on multidisciplinary fundamental education, combining, along with biological disciplines, serious training in chemistry, physical chemistry, molecular physics, computer science and bioinformatics. To do this, it is necessary to create original special courses, special practices, master's degree programs.

The experience of Lomonosov Moscow State University, MIPT and a number of scientific and educational centers can be used here. So, at the Faculty of Biology of Moscow State University within the framework of the National project "Education"  An innovative master's program "bioengineer-manager" has been developed.

One of the tasks of the bioengineer – manager project is to develop a mechanism for training personnel who are at the forefront of scientific and technological progress and are able to fill the existing personnel deficit. This Master's program aims to train personnel for the organization of research and innovation process in the most rapidly developing fields of biology, bioengineering, biotechnology and nanobiotechnology.

At the same time, it should be borne in mind that the master's degree is only the first step in the formation of a highly qualified specialist. In two years of training, it is impossible to invest in it all that is needed in modern bioengineering and nanobiotechnology. Therefore, it is important to teach the future specialist to study both modern scientific achievements in the chosen field, and to give him organizational and economic skills. For this purpose, within the framework of the subproject, a fairly extensive pool of special courses and special practices focused on achieving this goal was developed.

It seems to us expedient to introduce a sufficiently flexible curriculum, within which the proportion of subjects chosen by the customer of the specialist would be increased. In general, given the serious material costs of the university in training specialists in the field of modern bioengineering and nanobiotechnology, it seems logical to train such specialists mainly in a targeted manner according to the orders of enterprises.

Conclusion. 
 
It is impossible to cover the problems of nanobiotechnology development even in the minimum necessary volume in one article. We have touched upon only a very limited range of issues lying, as they say, on the surface. However, it is already clear how big and large-scale the consequences of progress in this area can be. There are more questions than answers right now. But the development of science in this area is very fast.

The role of fundamental science in understanding the general patterns of interaction of artificial nanostructures with biological objects will be great. An innovative business that accumulates scientific achievements and translates them into the necessary end products will be of great importance.

The role of education and training for both basic research and innovative business cannot be overestimated, especially in the near future. And it is already clear that very serious work is to be done in this direction. The development of nanotechnology and, in particular, nanobiotechnology will undoubtedly be a powerful locomotive of all progress in the coming decades.

About the authors:Kirpichnikov Mikhail Petrovich, Doctor of Biological Sciences, Academician, member of the Presidium of the Russian Academy of Sciences, Dean of the Faculty of Biology of Moscow State University, Head of the Department of Bioengineering.

• Shaitan Konstantin Voldemarovich, Doctor of Physical and Mathematical Sciences, Professor, Deputy.Head of the Department of Bioengineering, Faculty of Biology, Moscow State University.
  
  

Portal "Eternal youth" www.vechnayamolodost.ru10.07.2008