And nuts are not simple…
Gold–plated nanosheets are a universal weapon in the fight against diseased cells
Vasily Yanchilin, "Search"
Nanotechnology is becoming more complex. For example, nanocomposites for the diagnosis of diseases and treatment are covered with complex organics – antibodies and reporter molecules. The basis for this is "spiked" nanostructures or nut-like nanowells. Their synthesis and further refinement is carried out by senior researcher, Candidate of Physical and Mathematical Sciences Vitaly Khanadeev from the Laboratory of Nanobiotechnology of the Institute of Biochemistry and Physiology of Plants and Microorganisms of the Russian Academy of Sciences (Saratov). His research, which is important for science and medicine, is supported by a grant from the President of the Russian Federation.
– In our nanobiotechnology laboratory, we study nanocomposites based on gold, silver, silicon dioxide, albumin. These are special structures about 100 nanometers in size, created using nanotechnology, which are then used to work with diseased tissues. Methods of "wet" chemistry are used for their synthesis, that is, the formation of nanoparticles occurs in solution. By adding metal salts, surfactants, reducing agents and controlling the synthesis parameters (temperature, pH), we obtain nanocomposites of various shapes, sizes and structures.
We are also able to cover nanocomposites with biocompatible polymers (polyethylene glycol, polyvinylpyrrolidone). Thanks to this "disguise", the immune system does not perceive them as foreign objects, and they circulate longer in the body. In addition, we can cover them with "recognizing" molecules, that is, antibodies that will selectively bind only to certain "target molecules". An example of such a "target" is the epidermal growth factor receptor (EGFR), which is found in large numbers on the surface of tumor cells. Thus, nanocomposites coated with EGFR antibodies will attach to the surface of tumor cells.
One of the most important properties of noble metal nanoparticles is the presence of plasmon resonance (PR), the essence of which is as follows. When a metal nanoparticle is irradiated with light and the frequency of the light wave coincides with the frequency of vibrations of metal electrons within the nanoparticle, a resonance is observed. As a result, it strongly absorbs or scatters light, that is, it heats up or glows.
These effects are widely used in biomedicine. For example, it is possible to destroy tumor cells by laser heating of nanoparticles attached to them (photothermal therapy). In this case, the intensity of laser irradiation is not very high, and it does not lead to damage to healthy cells. However, it causes a strong heating of metal nanoparticles and the death of tumor cells to which they have joined.
It is also possible to "see" tumor cells due to the scattering of light from attached nanoparticles. Cell imaging is usually used for diagnosis, but it can also be used for treatment (for example, during surgery to remove a tumor, a surgeon will be able to notice "glowing" metastases and cut them out when illuminated with a laser).
In recent years, PR-nanostructures have been frequently used for the study of various substances using Raman spectroscopy (RAMAN). The effect of Raman light is that fluctuations in chemical bonds in the substance under study make a small contribution to the change in the spectrum of scattered light.
A significant increase in the RAMAN when the molecules of the substance under study are attached to the metal surface is called giant raman scattering (GCR). It was found that the maximum field amplification is observed when the molecules are located inside thin gaps (several nanometers thick) and near sharp protrusions. These areas on the metal boundary are called "hot spots". Creating nanocomposites with the maximum number of such "hot spots" is just one of the tasks of our project.
One of the promising directions for the development of GCR applications has become the use of so-called GCR tags – metal nanoparticles coated with reporter molecules of the KR. Reporter molecules have characteristic RAMAN spectra, and metal nanoparticles significantly amplify the signal from them. Such labels have great potential for use in biomedicine and, in particular, in visualization.
GCR tags based on multilayer nanoparticles have noticeable advantages due to the location of reporter molecules in the gaps between the metal layers. The field in these gaps is much stronger than on the surface, and therefore the RAMAN signal from the molecules located there is more intense. Also, the reporter molecules are protected from external influences and cannot detach from the particles. We plan to create such multilayer GCR tags based on gold nanostars.
– What are nanostars and nanowells?
– Nanostars are balls covered with sharp spikes. The "embryos" for their synthesis are gold nanoparticles, on which spikes are formed due to the rapid recovery of gold. By changing the size of the "embryos" and their concentration, we can adjust the size of the final nanostars, the length and number of spikes. The size of nanostars can range from several tens to several hundred nanometers.
Photo provided by V.Khanadeev
Gold nanowells are silicon dioxide balls covered with a thin gold layer. By the way, their prototype is mentioned in Pushkin's "The Tale of Tsar Saltan": "And the nuts are not simple, all the shells are golden." The size of the silicon dioxide core is usually from several tens to several hundred nanometers, the thickness of the gold coating is from ten to fifty nanometers.
The interest in these types of nanoparticles is caused by their unique optical properties – intense plasmon resonance and the ability to adjust it to a given wavelength by changing particle parameters (beam length and core size in nanostars and the ratio of core and shell sizes in nanowells), which we can adjust.
For raman scattering, it is important that the electric field will be intense near the sharp spikes of nanostars and stellate nanowells and we will be able to measure the amplification of the RAMAN signal from the molecules placed there. Moreover, we are going to cover the nanostars with several layers of silver shell. In the thin gaps between them, a sharp increase in the field will be observed, which will lead to an amplification of the RAMAN signal from the molecules located there.
If we estimate the GCR signal for particles of the same size, then golden nanostars have the highest gain of the RAMAN signal compared to nanowells and other particles. However, for nanostars with a core size of more than 100 nm, their mass increases significantly, and they quickly settle to the bottom from the solution. To eliminate this disadvantage, the synthesis of stellate nanowells, which have golden rays on their surface, seems promising. The advantage of nanowells is that they have a small mass due to a light core with a sufficiently large size. Now it is difficult to say which of these nanoparticles will be the most promising. To find out this and study their properties in more detail is just our task.
– Do you study nanocomposites and develop them?
– Yes, we are not only researching, but also participating in their development. Now this direction is developing rapidly, and we are also making our contribution. In addition, the accumulated experience and tracking of new trends allow us to significantly optimize the known synthesis methods.
The research process includes several stages. First – search for literary data on the chosen topic, so as not to reinvent the wheel and make sure that the research is relevant. Then – experiment planning, synthesis, control of the shape and structure of nanocomposites.
Using electron microscopy, we get images of particles and can find out which substance corresponds to each point in the image. It is important to control the optical properties of nanoparticles – the wavelength and intensity of the plasmon resonance, as well as the amplification of the Raman signal. Sometimes we perform theoretical calculations to better understand the dependence of optical properties on the shape and size of nanoparticles. After passing these stages, we test nanocomposites on animal cells, including cancer cells, to make sure that there is no toxicity and to evaluate the effectiveness of their use. Then we turn to animal experiments.
– In what field of biomedicine can your nanocomposites be used?
– Synthesized nanocomposites can be used in several areas of biomedicine at once. Stellate nanowells will act as biosensors inside a living organism. By amplifying the RAMAN signal from surrounding molecules located near the spikes, we will be able to get information about the chemical composition of the environment and learn about the processes taking place there.
We will cover the nanostars with several layers of silver and embed the reporter molecules into the gaps between the layers. We will use them as labels for visualization due to the RAMAN from reporter molecules located inside the gaps, that is, in the area of "hot spots".
In addition, the outer silver surface can be used to attach "recognizing" molecules during targeted delivery of composites to "target molecules", for example, to cancer cells, as well as for subsequent photothermal therapy. Since we plan to obtain nanocomposites with maximum amplification of the RAMAN signal and with pronounced plasmon resonance, their administered dose and laser irradiation time will be reduced, which will reduce side effects.
– Is it possible to compare your research with foreign ones?
– The level of our work corresponds to the world. This area is developing very quickly, and we are also involved in this. In particular, scientists synthesized nanostars for the first time ten years ago, began to cover them with silver to enhance the RAMAN signal four years ago, to include reporter molecules between the layers – three years ago.
In the course of research, it was found that the formation of several layers of metal, with reporter molecules enclosed between them, can lead to even greater signal amplification – then the active synthesis of the so-called "nanomatracks" began. By the way, we also took part in their development. In 2015-2016, our joint Russian-Chinese project was dedicated to this topic, which was simultaneously supported by the funds of the two countries.
Nanostars with multiple layers of silver will be synthesized for the first time. We will check at which particle parameters the RAMAN signal from the molecules will be maximally amplified. It is not necessary to apply many layers, since too thick a silver shell can weaken the signal.
– What result do you expect to get?
– We plan to synthesize the nanocomposites I have described and determine their optimal dimensional parameters (thickness and number of layers of silver shell in nanostars, core diameter, shell thickness and ray size in stellar nanowells) to obtain maximum gain of the GCR signal. We will also conduct a theoretical study of the local amplification of the electric field inside the cavities and near the surface of nanocomposites under the action of laser radiation. In addition, we will test nanocomposites on biological models – cancer cell lines – and on vaccinated cancer tumors in laboratory animals.
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