Nanomacons for theranostics
B MIPT came up with a smart material for express DNA diagnostics
For more than a hundred years, humanity has been trying to create a "magic bullet", the concept of which was first proposed by the German doctor Paul Ehrlich. The idea is simple: "smart" particles are injected into the body, which themselves find, recognize and immediately treat the disease. Scientists are still struggling to implement this bold idea.
In the laboratory of nanobiotechnology The Moscow Institute of Physics and Technology, together with researchers from the Institute of General Physics of the Russian Academy of Sciences, have advanced especially far in solving this problem. In a recent paper published in the journal ACS Nano (Cherkasov et al., Nanoparticle Beacons: Supersensitive Smart Materials with On/Off-Switchable Affinity to Biomedical Targets), a group of Russian authors (without foreign affiliations) Under the leadership of Maxim Nikitin, MIPT presented a "smart" material unique in its properties, which can be used both for express DNA analysis and for creating a new generation of cancer and other complex diseases.
The delivery of drugs to the affected cells of the body is currently a weak link (a narrow "bottleneck") of diagnosis and therapy. Ideally, the drug should be targeted – only to "sick" cells, without causing any harm to healthy ones. It is possible to distinguish a diseased (for example, cancer) cell from a healthy one by various compounds (markers) on its surface or in its microenvironment – waste products or various signals transmitted to other cells of the body.
Existing drugs isolate diseased cells by one such marker. However, almost always there are markers of a diseased cell on healthy cells, only in smaller quantities. That is why the existing systems of targeted delivery are imperfect.
To increase the specificity of drug delivery, "smart" materials are needed that are able to analyze several parameters of their environment at once and more accurately find a target. "Generally accepted methods of drug delivery resemble a letter indicating the city and street, but without the house and apartment number," comments Maxim Nikitin, the head of the study. – For effective delivery, you need to be able to analyze more parameters." In 2014, in the journal Nature Nanotechnology, Maxim Nikitin and his co-authors published the results of a work in which they for the first time endowed nano- and microparticles with the function of performing any logical calculations using biochemical reactions. Such autonomous nanocomputers are able to identify the target much better by analyzing many of its parameters.
Despite the large amount of effort spent by many research groups around the world to expand the functionality of such materials, their main weak point remained low sensitivity to disease markers, which made it impossible to plan their actual applications.
In the current work, Russian scientists have managed to make a breakthrough. They have developed a unique smart material that has hypersensitivity to DNA signals, not just several orders of magnitude higher than the sensitivity of all other materials, but also better than the absolute majority of existing rapid DNA tests.
To achieve this outstanding result, the researchers were helped by the phenomenon of unusual behavior of DNA molecules on the surface of nanoparticles.
In the process, the authors sewed a single-stranded DNA molecule with one end onto the surface of the nanoparticles. It is important that this DNA molecule did not have double-stranded regions formed due to the pairing of fragments of its own chain (the so-called "hairpins"). A receptor was sewn onto the other end of the DNA strand, recognizing markers on the cell surface. To the surprise of the researchers, the receptor did not want to bind to the target in any way. And it wasn't a mistake. There was a hypothesis that on the surface of the nanoparticle, a single-stranded DNA strand "sticks" to the surface and spontaneously curls into a ball, as a result of which the receptor "hides" on the surface of the nanoparticle (see Figure 1). The hypothesis was confirmed when another small DNA strand complementary to the DNA on the nanoparticle was added to such a particle – the receptor was instantly "activated" and bound to the target. Due to the formation of complementary pairs between the nucleotides, the two strands formed a rigid double helix, or, as scientists say, a duplex. As a result, the DNA strand, like a chameleon's tongue, unfolded, and the receptor began to recognize the cellular marker.
Drawing 1. Activation of a receptor on the surface of nanoparticles when a complementary DNA strand is added. Provided by the authors of the study.
In behavior, this design resembles the well-known in science "molecular beacons" (see Figure 2). Their principle of operation is that the "hairpin" is responsible for folding /unfolding, that is, the formation of a duplex single-stranded DNA strand with the other end at one end. Competing for binding to the ends of the beacon, complementary DNA can break the hairpin and unfold the DNA of the beacon.
In the phenomenon with a nanoparticle, a fundamental and extremely useful difference is achieved.
"This is a unique artificial interface that makes the folding force of DNA (the interaction of DNA with the surface) and the force that unfolds it (the formation of a duplex) independent. Due to this separation, we dramatically improve sensitivity," says the first author of the article, Vladimir Cherkasov, a leading specialist in the laboratory of nanobiotechnology at MIPT.
Drawing 2. Comparison of molecular beacons and smart materials developed by the authors of the study. Provided by the authors of the study.
In the published article, the authors demonstrated agents capable of detecting DNA in concentrations up to 30 femtomoles per liter (femto = 10-15) without DNA amplification and/or signal. Elizaveta Mochalova, a co-author of the work, a graduate student of the Laboratory of Nanobiotechnology at MIPT, explains:
"This sensitivity was demonstrated in an extremely easy-to-use immunochromatographic analysis, known in the format of a pregnancy test. Such tests can be carried out without the use of clean laboratory facilities and sophisticated equipment, usually necessary for existing DNA analysis technologies. This technology is suitable for rapid screening of infectious diseases, home food analysis and the like."
In addition, the authors showed the applicability of the technology to create smart nanoagents for the recognition of cancer cells depending on the content of small DNA in their microenvironment. Although until recently it was believed that small nucleic acids are meaningless "scraps" of large functional molecules, now it has become clear that small RNAs are key regulators of many processes in living cells. Currently, there is an active identification of similar RNAs that are markers of diseases.
Maxim Nikitin, Head of the MIPT Nanobiotechnology Laboratory, comments:
"Interestingly, the shorter the length of the detected nucleic acids, the fewer competitors this technology has. Up to the point that at the moment there are no agents that would be able to detect sequences of 6-9 bases with such sensitivity. We can already create agents controlled by well-studied small RNAs (17-25 bases long). But even more exciting is that our method allows us to probe the microenvironment of cells for the first time and understand whether small RNAs of shorter length are useful indicators of diseases, and not "garbage", as is still considered due to the difficulties of their detection."
The developed technology opens up new prospects in the field of genomic technologies – both from the point of view of express DNA diagnostics outside laboratory conditions (at home, in the "field", etc.), and for the construction of therapeutic nanomaterials of a new generation. Indeed, in recent years, colossal breakthroughs have been made in the study of the genome and its editing, but this technology can solve the still urgent problem of drug delivery to cells only with a certain genetic profile of the microenvironment.
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