Nanobiotechnology: a systematic approach to living

The latest branches of biotechnology will allow solving many social and economic problems facing modern society in the future. This area of research can become a reliable source of patentable technologies that bring profit to states and private companies. It will contribute to the development of scientific and technical potential that ensures the health of the nation, its biosafety, control over the spread of infectious and somatic diseases. Biotechnologies are the tool with which fundamentally new personalized medicines can be developed, allowing, as Hippocrates once declared, to treat not the disease, but the patient.

That is why developed countries, experiencing another technological – already nano–technological - revolution, pay special attention to the development of biotech.

Professor Vadim Govorun, Deputy Director for Scientific Work of the State Research Institute of Physico-Chemical Medicine of Roszdrav, tells about what is happening in the rapidly developing field of nanobiotechnology.

The interview was recorded by Vladimir Sychev, STRF.ru , for the "Russian Electronic Nanojournal".

– Today we are witnessing the rapid development of biotechnologies, going in several directions at once.

Firstly, technologies for determining the structure of biopolymers have been significantly improved. It turns out that it is possible to "read" and analyze biological texts (determining the nucleotide sequence of DNA, establishing the amino acid sequence of proteins). This has made it possible to almost completely decipher the genetic information contained in the human genome, as well as in the genomes of the main pathogenic and many industrially significant microorganisms and viruses (producers, vector systems, etc.). Therefore, unique prerequisites are being created for the development of new technologies for the treatment and prevention of diseases. In the foreseeable future, it will be possible to talk about the creation of personalized medicine.

Secondly, the informatization of research allows, in essence, to talk about the transition from medical empiricism to pragmatism, from sorting through a variety of medicinal compounds during experiments to purposefully creating compounds with predetermined properties. Now it is possible to invent and produce new types of therapeutic agents in silico.

Finally, another fundamentally important feature is the miniaturization of devices and materials used in biomedical research. It becomes possible to simultaneously measure a large number of parameters of the studied objects.

There is a gradual transition of research from the microcosm to the nanoscale, to the scales characteristic of the sizes of individual molecules. Ultimately, due to the reduction in the size of measuring devices, it will be possible to determine not the concentration of molecules in the sample under study, but their number.

In general, we can talk about the emergence of not only new knowledge and skills, but also a whole field – nanobiotechnology. Specialists working in it use the fundamental knowledge accumulated in previous periods of the development of science to construct analogues of living objects or their parts and to give them properties comparable or superior in their characteristics to living systems.

Nanobiotechnology is an interdisciplinary field, but its main component is medical. This includes the creation of new diagnostic and control systems, necessary, for example, for adequate, moreover, personalized therapy; and the development of new drug compounds and targeted drug delivery systems.

The creation of new biocompatible materials with which it will be possible to replace damaged tissues and organs is also becoming a reality. Therefore, one of the main goals of nanobiotechnology is to copy known, studied macromolecules and molecular complexes or their functions, which will make it possible to compensate for defects that accumulate during the life of a complex biological system. Biostructures that have served their time can be replaced with artificial ones. It will be possible to selectively remove pathologically altered biostructures to prevent the processes of degradation, malignancy and obstruction of organs and tissues.

Nano-bio-tech: three ways– There are three main directions of development of modern nanobiotechnologies.

The first, nanobiotechnology of living systems, implies giving living systems (primarily microorganisms) by directed modification of the properties necessary to provide a certain function (or even a technological cycle when creating completely artificial nanoconstructions). The same direction includes the use of microorganisms as producers of nanomaterials.The second direction is "semi–synthetic" nanobiotechnology.

Here we are talking about the use of biopolymers: proteins, nucleic acids, other molecules and their complexes to create various nanobiotechnological devices (biomotors, pores, sensors). Further, using the principles of self-assembly or synthesis of organic and inorganic molecules, devices can be created that perform strictly defined functions of the copied biological structure. It is also possible to create biocomputers based on the processes of self-assembly of macromolecules. Such biocomputers can be used to diagnose diseases.

Finally, the third direction is "synthetic" nanobiotechnologies, the forerunners of technologies for creating devices designed to correct molecular errors and primary diagnostics of the state of the body, tissues, cells. Here it is supposed to use the phenomenon of self-assembly or synthesis of organic and inorganic molecules to create devices from numerous atoms ordered relative to each other.

Live Electronics– If we talk in more detail about the use of living objects for nanobiotechnological purposes, then first of all it is necessary to mention the technology of obtaining various nanoparticles (magnetic, quantum dots and others) in natural bioreactors – bacterial cells.

For example, the cells of magnetotactyl bacteria Magnetospirillum magneticum can synthesize magnetite particles – Fe 3 O 4, and, curiously, the size of the nanoparticles depends on the conditions of bacterial cultivation. It is also important that such "production" of bacterial cells is surrounded by a membrane, so magnetite particles are easily isolated from the solution. By now, the sequences of Magnetospirillum magneticum genes responsible for the synthesis of nanoparticles have already been determined, so that using genetic engineering methods, it is possible to directly influence the parameters of the resulting nanoparticles.

Such particles can be used in a variety of methods: for example, in diagnostics using immunochemistry, in cell separation systems – cell separation, nucleic acid isolation, control of targeted drug delivery, local hyperthermia. In addition, it is possible to use such nanoparticles for the purposes of atomic force microscopy.

This is not the only example of nanoparticle synthesis in vivo. It is now possible to obtain nanoparticles consisting of metals such as cadmium and lanthanum by bacterial synthesis (which, in turn, can provide a breakthrough in the technology of creating components of microelectronic devices – approx. STRF.ru ). Yeast cells are also used as a nanobioreactor. For example, in the yeast cells of Schizosaccharomyces pombe, metal-peptide complexes are formed – microcrystallines with a size of slightly less than two nanometers, actual quantum dots that can be used in semiconductor devices.

Finally, the synthesis of nanostructures can also occur in solution using components of the bacterial cell wall. In this case, we are talking about the use of protein molecules that make up the so-called S–layers - regular structures on the surface of bacteria. The results of work on the creation of in vitro membranes consisting of S-layer proteins and having a controlled pore size have already been described. It is noteworthy that such structures may include "guest" molecules embedded inside the pores.

Of course, nanobiotechnologists use not only individual molecules, but also large molecular ensembles (for example, viruses) formed by self-assembly. A classic example is the tobacco mosaic virus, which has long been one of the favorite objects of virologists. It is a symmetrical rod-shaped protein capsid (cylinder) consisting of more than two thousand identical protein molecules arranged in a spiral. Inside the viral capsid there is a cavity in which a ribonucleic acid molecule is placed. Such viral structures can be used as a nanocontainer for other nanoparticles, primarily metals; used as a matrix (or "skeleton") for forming on their surface by decorating metal nanowires. As a result, they can serve as nanoelectrodes and find application in microelectronics.

And how are new technologies used to deliver medicinal compounds?– Here we are talking about vesicular nanosystems – these are particles (liposomes, micelles) or molecules (fullerenes and dendrimers).

Micelles and liposomes are the most well studied, they have been used for solving applied and fundamental problems for quite a long time. Stabilized micelles of 5-50 microns in size, consisting of natural or artificial phospholipids, are used as means of delivering medicinal compounds to target cells. Such particles have high solubility and easily penetrate histo-hematic barriers, making it possible to deliver drugs to various tissues and organs. Sometimes the structural blocks used for the production of micelles have a pronounced therapeutic (antibacterial, antiviral and fungicidal) effect and, therefore, can be used as a new generation of antibiotics.

The use of nanostructures for drug delivery seems very promising. However, there are concerns that the use of new techniques will be associated with a risk to human health…– Of course, the development of new algorithms for testing this kind of drugs is required, because unlike diagnostics (which at least takes place outside the human body), no one has thoroughly studied safety-related aspects in relation to new generation medicines.

Perhaps these are unnecessary worries and fears. But it may be that the use of nanoparticles, dendrimers, and other carriers of medicinal agents can cause serious complications. So far, there is no metrological or pharmacological base to test all this.

A motor made of moleculesLet's move on to "semi-synthetic" biology.

What can we say about biopolymers as a kind of "building blocks" for new devices?– From this point of view, two main classes of biopolymers are most important – proteins and nucleic acids.

Proteins are interesting primarily because they are the main components of molecular machines, the structure and functions of which have been "honed" during hundreds of millions of years of evolution. As an example of "molecular motors", it is necessary to cite ATP synthase and flagellar flagella of bacteria that provide rotational movement. Translational, linear motion is the prerogative of proteins such as myosin and kinesin. And researchers already know how to "turn on" and "turn off" the operation of such motors, for example, the same bacterial flagella.

As for nucleic acids, their main feature associated with functioning both in living cells and in nanobiotechnological applications is the ability to mutual recognition, complementary interactions of their chains. Indeed, the attractiveness of nucleic acid molecules lies, among other things, in a high degree of complementarity: the limit of mutual recognition of DNA chains is three angstroms – this is a very high accuracy.

The principle of complementarity is fundamental for the creation of nanoconstructions based on nucleic acid molecules. DNA molecules or their fragments can be used as a building material for nanofabrics of the future. Such "building blocks" will allow not only to create planar structures of a certain shape and size, but also to proceed to the design and creation of volumetric nanoconstructions.

Diagnostics using a biocomputerDiagnostics is one of those areas where very serious changes are expected with the development of biotechnologies…

– Generally speaking, diagnostics in a broad sense is an integral part of any technological process – whether it is the assembly of nanodevices, the treatment of patients, the creation of medicines.

It is not surprising that the miniaturization of diagnostic devices and the appearance of new properties in them – multiparametricity, led to a steady increase in the interest of the diagnostics market in nanotechnology. In the process of creating new methods and devices designed for diagnostics, related technologies are also involved – microfluidic technologies, micro- and nanoelectronics, probe microscopy, the technique of spectral analysis of single molecules.

What will be the specific applications of the new detection technologies?– Perhaps the most typical example is the creation of new technologies for sequencing DNA molecules.

Fast, economical and, most importantly, reliable ways to determine the sequences of nucleotides in their composition are needed by both physicians and specialists in genotyping organisms, criminologists and so on.

The currently existing methods of sequencing DNA molecules have a disadvantage due to the fact that at a certain stage it is required to amplify, "multiply" nucleic acid molecules using a polymerase chain reaction. But the fact is that the DNA polymerase enzyme, which synthesizes new DNA molecule chains, makes mistakes that accumulate during amplification. How to get around these difficulties? Obviously, it was necessary to develop direct, non-chemical methods for decoding the nucleotide structure of DNA molecules.

Among the new techniques, sequencing using nanopores should be mentioned first of all. The concept of using small–sized holes – pores - was developed at Coulter, which uses pores to count particles from submicron to millimeter size. Registration of DNA molecules occurs as follows. The molecules are suspended in an electrolyte solution divided into two reservoirs. A preset voltage is applied to the walls of the channel connecting the tanks, and the nucleic acid molecules begin to pass through the channel. When a single molecule enters the channel, the electrical resistance increases in it, and each new molecule is registered by changing the current.

Currently, there are two approaches: the use of pores from protein molecules (for example, alpha-hemolysin) and the creation of inorganic pores with a long lifetime. The main goal of the developers of new technologies for sequencing DNA molecules (it has not yet been achieved) is to learn how to recognize individual nucleotides in the DNA or RNA molecule. The idea of the researchers is that the electrical characteristics of different nucleotides will differ when passing through the channel. It is clear that it is necessary to minimize the channel length to ensure high resolution. However, it is impossible to achieve the necessary stability of the channel with existing technological techniques. This development will require new efforts – in particular, the creation of methods for the formation of pores in ultrathin (two to three nanometers thick) films.

The next area of diagnostics is nanoproteomics. Actually, the term "nanoproteomics" was first proposed by our compatriot, Professor Alexander Archakov, it meant using the method of atomic force microscopy to identify individual protein molecules or their complexes. According to the ideas of Alexander Archakov and co-authors, molecular diagnostics should be associated with the determination of single protein molecules, and not their concentration in the test sample. Indeed, there is a fundamental possibility of visualizing not only protein complexes, but also individual macromolecules on a substrate. And several companies are developing multichannel atomic force microscopes and special modified substrates for the detection of viruses, bacteria, toxins and antigens. The main disadvantage of this approach is the lack of specific features of the resulting image, which sometimes dramatically affects the accuracy or specificity in determining the analyte. But the technique of microscopy also does not stand still, along with the improvement of tunneling microscopy methods, other areas of nanoproteomics are actively developing, designed to solve research and medical problems.

Another area of application of nanoproteomic technologies is the separation of mixtures of proteins and peptides with subsequent mass spectrometric analysis. Metal nanoparticles (aluminum oxide, silicon) and synthetic nanoparticles are used in the study of separation processes of model mixtures of peptides and proteins. According to the authors of these papers, nanochromatography significantly, almost by an order of magnitude, improves separation and allows for better results of proteomic analysis for the identification of peptides and whole proteins during further mass spectrometric analysis.

Detection systems are becoming more and more miniaturized. Probably, over time, it will be possible to create devices capable of determining the parameters of the vital activity of even a single cell?– Today it is impossible, but many technological elements that are used to create modern diagnostic systems already look like real prototypes of future nanodevices intended for practice.

For example, micro- or nanofluidic bioanalytical systems combining elements of electronics, micromechanics, optics and hydraulics. The basis of such systems is a glass or polymer plate with a multilevel system of channels, microreactors, valves and pumps, operating with micro-, nano- and femtoliter volumes of liquid. Microfluidic systems allow working with individual cells at different stages of their development. Radical miniaturization of the sizes of experimental devices achieved using micro- and nanofluidic technologies makes it possible to switch to qualitatively new, less expensive methods for solving a wide range of fundamental and applied problems of molecular and cellular biology, biotechnology and biomedicine.

What do "nano-" in "bio-"And what about materials like fullerenes and nanotubes?

What is their place in the field of "nanobio"?– They are nanocontainers for various organic compounds exhibiting antiviral, anticancer and antibacterial activity.

I mentioned above that micellar systems with modified phospholipids can be used as a new generation of antibacterial drugs. Similar antibacterial, anticancer activity is also characteristic of fullerenes. For example, fullerene C 60 has been used to treat viral infection and cancer in animals.

The unique properties of fullerenes are due to their high reactivity due to the large number of free carbon valences. For use in biomedicine, pure fullerenes are of little use due to their insolubility in aqueous solutions and, as a consequence, restrictions on the concentrations used in the study of their properties on animals. However, the functionalization of fullerenes (for example, the production of carboxyfullerenes) makes these compounds bioavailable and, therefore, more effective for research in biosystems. One of the ways to introduce fullerenes into the body is encapsulation in a lipid vesicle for targeted delivery to transformed cells. The use of the principles of photodynamic therapy and the generation of singlet oxygen by fullerene under the action of light causes damage and death of the target cell.

Nevertheless, the main directions of using carbon nanotubes in biology and medicine are associated with their unique mechanical and electrical properties. Technologies of immobilization of enzymes and even enzymatic complexes, which are the analytical element of the nanobiosensor on the inner and outer sides of the nanotube, have already been mastered. The work of the enzyme with a certain substrate (for example, the use of glucose oxidase) allows you to create a highly sensitive sensor that measures the concentration of glucose. Various biochemical sensors for the determination of toxic substances, cations and anions, antigens of pathogenic viruses and bacteria, as well as prion proteins are described. Nanotubes are used to provide targeted delivery of medicinal compounds, macromolecules (proteins, DNA) to target cells. And the combination of nanotubes with nanoparticles made of metal oxides serves as a substrate in cell cultivation and the creation of prototypes of organs and tissues.

In addition, bionosensors using functionalized nanotubes and metal nanowires are being actively developed. Such projects involve the immobilization of sensor molecules (enzymes, antibodies, lectins, etc.) to create a multiparameter biosensor compatible with body tissues or cells in culture.

Technologies of the near futureWhat conditions do you think will contribute to the development of nanobiotechnology in Russia in the near future?

– The rapid development of nanobiotechnology and related nanotechnology disciplines leads to the involvement of new research groups and entire institutes in these research programs.

Most nanobiotechnological projects are currently at the stage of initiation or obtaining the first results. However, the tools, ideology and technology of "nanobio" have already been formed. As is often the case in modern technological disciplines, the processes taking place in different laboratories and companies, interacting with each other, will begin to give the first practical results in the near future. The appearance on the commercial biotechnological market of new systems for determining the nucleotide sequence of DNA, nanodosators, microfluidic laboratories marks the gradual transition of biotechnologies to another research format and will inevitably give a different quality of the results obtained.

It is worth emphasizing that the complexity of technologies, their complexity, require clear cooperation of various research groups to achieve this goal. Nanobiotechnology, I repeat, is a multidisciplinary science, the participation of a single research laboratory in this process is ineffective. In fact, we are talking about creating technological platforms – sets of ideas, competent specialists, material and technical equipment that operate at the junction of different disciplines. Therefore, for example, it is no coincidence that teams that develop the concept of systemic or synthetic biology participate in nanobiotechnological projects. Ultimately, the volume and quality of knowledge accumulated in the course of systemic biological research will determine progress in nanobiotechnology.

The interview is published in abridgment with the permission of the "Russian Electronic Nanojournal" (read the material in full on the magazine's website).

Portal "Eternal youth" www.vechnayamolodost.ru26.05.2008