Available online on 15.03.2024 at http://jddtonline.info

Journal of Drug Delivery and Therapeutics

Open Access to Pharmaceutical and Medical Research

Copyright  © 2024 The   Author(s): This is an open-access article distributed under the terms of the CC BY-NC 4.0 which permits unrestricted use, distribution, and reproduction in any medium for non-commercial use provided the original author and source are credited

Open Access  Full Text Article                                                                                                                                                      Research Article

Development of a technique for determining the viability of dormant organisms in powdered substances

Ivan A. Gaidashev *, Yuliana G. Nikolaeva, Anton V. Syroeshkin

Department of Pharmaceutical and Toxicological Chemistry, People's Friendship University of Russia (RUDN University), 6 Miklukho Maklaya St., Moscow, 117198, Russian Federation

INTRODUCTION

Monitoring the viability of pharmaceutical substances manufactured on a bacterial, plant or animal basis has the following main purposes:

- Preserving the viability of bacterial cultures in the lyophilized state, for example, to maintain the stability of the digestive system ¹;

- Absence of live cells in dried medicinal plants for their stability at storage ²;

- Absence of active or potentially active life forms to prevent infection (prions ³, viral ⁴ or bacterial ⁵ forms, or invasions ⁶).

Rapid determination of the presence of alive things is a non-trivial scientific task. The presence of alive things is always associated with slow conformational rearrangements of oligomeric intracellular complexes ⁷. Slow conformational transitions are one of the main characteristics of oligomeric proteins and their complexes with membranes and other biopolymers as a result of the multiplicity of their conformational states that depend on the ligand environment, electric field strength on the biomembrane, temperature, and many other factors. Besides the multipoint Coulomb binding, which is well known for low-molecular substances, biopolymers ^8-10 interact with a supramolecular target due to dispersion (Deryagin) "surface-to-surface" interactions ¹¹. A feature of these interactions is the Coulomb tuning of the flickering dipoles due to the mutual electromagnetic radiation of the surfaces. This interaction is dynamic, a mutual influence of quantum fluctuations on the electron density distribution occurs in complex matrices ¹². The contribution of dispersion interactions to the free energy of the adhesion reaction of surfaces is an order of magnitude larger than the interaction of single charges ¹³. Therefore, the dynamic redistribution of dipoles on the surfaces, when a supramolecular therapeutic substance is binding to a supramolecular receptor, is a critical condition in the mechanism of the biological activity of nanoparticles. It would be expected that the electromagnetic emission of flickering dipoles can mimic the effect of a ligand on an enzyme, and then ligand-specific conformational transitions of supramolecular complexes can be induced by mimicking the effect of low- and high-molecular substances under changing thermal fields and emissions. Thus, thermal radio emission of macromolecules ¹⁴ can be used as a catalyst in biological reactions, as well as as an active substance that causes a change in the release of secondary messengers in the chain of ligand-receptor interaction.

An advantageous model for studying conformational transitions in macromolecules are the dormant eggs of the brachiopod crustacean A. salina because of their ability to maintain viability under extremely unfavorable environmental conditions. Dormant eggs have the ability to fast hydrating-dehydrating, which is an extremely important factor in primary (molecular) stability ¹⁵. Fast dehydrating increases the viscosity in the cell and preserves non-freezing water, which has a beneficial effect on the low temperature tolerance, since there is no crystallization or mechanical damage to the cell ¹⁶.

Under unfavorable conditions, with small amounts of water in the cell, a significant decrease in the conformational mobility of high-molecular compounds occurs, which leads to a decrease in the degradation of the molecular structural-functional components of the system ^17-18. Conformational mobility is regulated by the balance of the interaction of water with polar groups of proteins and the hydrophobic interactions of the lipophilic parts of protein molecules. Drying proteins under vacuum results in a significant loss of water, however, it often does not lead to irreversible changes in the protein structure. Using the lyophilization method shows a more complete removal of water, which can lead to irreversible changes in their structure. Thus, the practice of preparing reference samples for trace element analysis using unstable and complex organic matrices widely uses sample stabilization by removing liquid through lyophilization. It is also noted that this procedure affects the organic matrix, which is due to irreversible changes in the conformation of proteins. It was shown that dormant eggs of A. salinae can remain viable even after drying by lyophilization ¹⁹.

This study proposes a rapid method for the viability control of powdered dry preparations. Lyophilized powder of Kalanchoe daigermontiana, dry dormant eggs of Artemia salina, and dry powder of Lycopodium sp. spores were proposed as model objects. Alive and inactivated preparations were characterized by elemental composition and intracellular water content. Suspensions of virus-like particles (VLPs) to SARS-CoV-2 were proposed as a cell-free reference material. It was shown that the listed model inactivated samples lost up to 90% of the density of their intrinsic flux of thermal radio emission in the millimeter band.

MATERIALS AND METHODS 

Objects of study

The following dormant forms were chosen as objects for studying thermally induced conformational transitions in the organic matrix: dormant eggs of the crustacean A. salinae, spores of Lycopodium sp., VLP SARS-CoV-2 vaccine, fresh shoots, shoot homogenate, and a reference sample made from K. dagermontiana. Samples of dormant eggs of A. salina, spores of Lycopodium sp., K. dagermontiana were tested for monodispersity ^20-22. Maximum size distribution: A. salina - 250 µm, Lycopodium sp. - 32 µm, reference sample of K. dagermontiana - 63 µm. Alive forms of A. salina and Lycopodium sp. germinated by 85% (dead forms - 0%).

Element analysis

Elemental content was monitored using two methods: XRF with the sample preparation technique used for making a reference sample based on K. daigremontiana and GZ-AAS as the reference method. Sample preparation of dormant eggs of A. salina using the GZ-AAS method was carried out in the same way as for a sample of the medicinal plant K. daigremontiana ²² using microwave digestion in Teflon bombs under pressure.

Determination of the flux density of thermal radio emission

The flux density of thermal radio emission in the microwave wavelength range was determined using a TES - 92 instrument (TES Electrical Electronic Corp., Taipei, Taiwan) with a sensor configured for anisotropic measurement along the Z axis. The measurement results are the maximum average flux density value with incremental averaging every 300 ms. The measurements were carried out both without opening the primary package and with opening the package. The preparations were heated to 37 ^оС using a solid-state thermostat with Peltier elements. The temperature of the samples was controlled by a remote laser infrared thermometer. The installation geometry did not change and was strictly observed for each sample. The spatiotemporal distribution of microwave background emission was controlled before each measurement with height, width, and length increments of 50 cm. The background radiation did not exceed 1 mW/m². Aqueous solutions were applied in drops of 100 ml to the center of sterile 10-cm Petri dishes. Drops were applied in the axial direction along the Z axis at a distance of 10 mm from the measuring sensor of the device. To assess the validation characteristics of in-lab precision, all measurements were performed at least seven times ²³.

 NMR spin echo technique

Instruments used in the study of proton spin-spin relaxation: XL-100 (Varian, Paolo Alto, California, USA), Avance 600 (Bruker, Billerica, Massachusetts, USA). The NMR instrument has several components ²⁴: 1) Computer for visualizing and averaging signal amplitudes and differentiating decay curves; 2) Unit that determines the sequence of 90^о and 180^о pulses to determine T₁, T₂; 3) Generator of radio frequency pulses that create a high-frequency magnetic field to rotate magnetic moments; 4) Oscillator circuit inductor, 0.4 T; 5) Oscilloscope – measures the spin echo signal and the transmitter signal. Temperature changes were provided by a unit with liquid nitrogen. The summation and accumulation of the results of individual measurements, the division of a multicomponent signal decay curve into individual components and the determination of T₁, T₂ for each component were carried out by a software specifically created for this installation. Nuclear magnetic resonance provides a wide range of parameters (shape, width, area, and position of the resonance line; time of spin-lattice T₁ and spin-spin relaxation T₂), that reflect the dynamics of molecules. The operating frequency of the installation is about 20 MHz. The measurement accuracy does not exceed 7%. The linearity of the installation's operation was continuously monitored using the aqueous reference solution of CuSO₄. NMR allowed obtaining information about changes in the conformation of organic macromolecules and mobile protons. The amplitude characteristics make it possible to determine the water content in the sample. Samples of A. salina dormant eggs were dried in a freeze-dryer. At the last stages of drying, the sample was heated to room temperature. For measurements, a loose sample of 0.5 g of dormant eggs was placed in a glass ampoule with a plug, and then the ampoule was placed between the poles of a magnet. After exposure of the sample to high-frequency pulses, the system of nuclear spins returns to equilibrium, that is, the nucleus transfers its energy to similar nuclei as a result of spin-exchange; this process of energy exchange between nuclei occurs according to the mechanism of spin-spin relaxation of nuclei (T₂). T₂ can be measured in the range from 10^-5 to 10² s. In our experiments, the components of immobile and low-mobility molecules were not recorded. A significant advantage of the NMR method is the possibility to study objects (including living ones) without violating their integrity or state.

RESULTS

Characteristics of live and inactivated samples of dormant eggs of A. salina

As is known, the state of water in the bulk phase, in the electrical double layer, and in the tightly bound state in crystalline hydrates and colloidal particles can be characterized by the spin-spin relaxation time (T₂) using the NMR-spin echo method ^{24, 25}: T₂ for column water molecules is 2 s, for loosely bound water –100-200 ms, for tightly bound water (not removed by drying – 5-15 ms, for ice – 50 μs.

To determine the kinetics of changes in content during storage in dormant eggs of A. salina, the NMR spin echo method was used. The data obtained illustrate a significant reduction, from 2.5 to 1 mg of H₂O per 1 g of biomass (Figure 1) when stored in a climatic chamber with a constant temperature of t=23 ^oC and humidity of 75%.

Figure 1: Change in the content of loosely bound water (T₂=220 ms) in dormant eggs of A. salina according to NMR spin echo data

Such slow drying does not affect the viability of dormant eggs, as it reflects the background metabolism of alive eggs frozen in the early gastrulation stage. 

Under accelerated drying (9 hours at 150 ^oC), dormant eggs lose their viability (that is, they die), and the water content drops to 0.5 mg/g.

The spin-spin relaxation time T₂ of protons in live dormant eggs under heating and that of the control sample of killed dormant eggs at 20^оC was 220 and 95 ms. Thus, the spin-spin relaxation time in killed dormant eggs significantly decreases, which indicates a change in the fraction of mobile protons and is confirmed by a decrease in water content (Table 1).

Table 1: T₂ proton relaxation time and total water content in samples of A. salina dormant eggs, alive and killed at 150^оС for 9 hours

It should be noted that the histological study detected no degenerative changes in the tissue.

Earlier, we proposed a method for distinguishing between live and killed dormant eggs based on the characteristics of elemental profiles obtained by the GZ AAS method under microwave digestion and XRF ²⁶. In this study, we used XRF to characterize the alive and killed homogenate and lyophilisate of Kalanchoe shoots.

Elemental analysis of K. daigremontiana shoots

We developed a reference sample from K. daigremontiana shoots for elemental analysis. The stability of indicators in the case of analysis by X-ray fluorescence spectroscopy of such a sample is ensured only in the absence of alive cells in the lyophilized powdered material. As part of monitoring the conformational transitions of the organic matrix using the XRF method, a prototype of a reference sample was developed using the following stages: 1) homogenization of the original substance; 2) lyophilization of the homogenate; 3) grinding the lyophilisate by direct knife blow; 4) sifting through a nylon sieve with a mesh diameter <63 µm. It is known that the complex organic matrix significantly influences the X-ray fluorescence yield. The sample preparation conducted allows for stabilizing the matrix and ensuring the preservation of repeatability and reproducibility over a long period of time. The key step in sample preparation is drying the sample. This study used freeze drying until the condensate flow completely stopped, which led to the removal of water retained by the electrical double layer and, as a consequence, a significant decrease in conformational mobility (increased stability over time) ²². Figure 2 shows the difference in the elemental profile of a live shoot homogenate and a shoot lyophilisate that does not contain live cells.

Figure 2: Elemental profile in fresh homogenate and dried sample of K. daigremontiana. Determined by XRF method, the element content was recalculated according to the protocol of the equipment manufacturer, Shimadzu. Black circles indicate the fresh sample, gray circles indicate the dried sample.

Thus, determining the elemental composition by XRF requires compliance with complex criteria to achieve the greatest reliability of the results. The elemental profile of the dried sample shows significant quantitative differences compared to the fresh K. daigremontiana shoot homogenate. The decrease in S content and a slight increase in Zn are due to the presence of sulfur-containing volatile compounds, which are removed by drying the sample, as well as a change in the localization of elements in the organic microenvironment in the case of zinc. The previous study showed that organic matrix stabilization by freeze-drying has the greatest effect on the increase in repeatability. Also, a stability test was performed on the reference sample for 210 days, the results of which showed a slight deviation in the content of elements from the original values.

To distinguish between live and killed medicinal plants, it is necessary to carry out long-term elemental analysis with complex sample preparation. For the samples of animal and plant origin presented in the first and second sections, we applied a new method of monitoring their own thermal radio emission, which allows rapid determination of their viability.

Rapid viability test for powdered substances

In a recent study, we showed that biologically active nanoparticles of irregular shape are capable of radio emission in the millimeter band ¹⁴. The flux density of this emission depends on the temperature, structural features of nanoparticles, their nativeness, and their concentration, which makes it possible to use this method for chemical-analytical control of infusions of medicinal plants and vaccines ²⁷. 

The conformational mobility of live and killed dormant eggs was proven by monitoring the thermal radio emission of dormant forms of powdered dormant eggs of A. salina, spores of Lycopodium sp, VLP-SARS-CoV-2 vaccine suspension, and fresh homogenized shoots of K. daigremontiana. When measured over 20 minutes, these samples showed a significant increase in thermal radio emission from 7 μW/m² to 35 μW/m², with background values <1 μW/m² (Figure 2). 

Figure 3: Thermal radio emission of VLP-SARS vaccine samples, powdered spores of Lycopodium sp., are indicated by a triangle, powdered dormant eggs of A. salina are indicated by a filled circle, and homogenized leaves of K. daigremontiana are indicated by a hollow circle.

Biologically active nanoparticles of plant or animal origin are isolated from living tissues and retain the properties of living things in an artificial environment that maintains them in their native state (buffer, given ionic strength, osmolality, limited temperature range). As is known, changing the storage conditions of isolated nanoparticles leads to their denaturation with minute kinetics. We assumed that nanoparticles of a live cell (oligomeric membrane and surface proteins, nucleoproteins, and peptides) would emit only as part of a live cell. Indeed, the flux density of thermal radio emission of powdered forms and suspensions drops by 3-10 times in killed samples (Table 3).

Table 3: Thermal radio emission flux density of live and killed dormant eggs of A. salina, native and denatured VLP-SARS-CoV-2 vaccine, freshly prepared homogenate, and lyophilized homogenate of K. dagermontiana shoots

1 - 100% corresponds to the emission flux density of 19 μW/m² from 1.0 g of A. salina dormant egg preparation at 37^оC.

2 - 100% emission corresponds to the emission flux density of 35 μW/m² from 0.1 ml of the VLP-SARS-CoV-2 preparation with a protein concentration of 160 μg/ml at 23^оC.

3 - 100% emission corresponds to the emission flux density of 7 μW/m² from 1.0 g of freshly prepared homogenate.

A lower flux density for the reference sample and killed dormant eggs indicates the cessation of the conformational activity of the supramolecular formations in the organic matrix and is another evidence of the significant influence of the sample preparation on the stability of the sample. Supramolecular emitting formations are apparently distributed in cellular structures that have absorptive capacity, which is leveled out by homogenization and release of these structures from the cellular matrix. This phenomenon may be species-specific due to structurally different supramolecular emitters. A significant change in the flux density of thermal radio emission in live dormant eggs indicates a change in the dielectric constant of the medium, which affects the yield of X-ray fluorescence caused by conformational transitions. No such increase was observed in killed dormant eggs.

DISCUSSION

It should be emphasized that XRF determination of elements in organic therapeutics is impossible without a reference sample: this is how international QA/QC conditions are met. Also, as part of the homogeneity test, a dispersion analysis was carried out using the LALLS method, showing the presence of a major fraction (by mass fraction) of about 65 µm and a minor fraction of about 39 µm. Stability tests of the prototype reference sample were carried out using the XRF method for 7 months, demonstrating less than 5% deviation from the original values.

A change in the organic matrix and, as a consequence, in the X-ray diffraction signal when heating the preparation from 25 ^oC to 37 ^oC, manifesting an effect similar to the heterogeneous catalyst, will make it possible to develop an enzyme-catalyzed synthesis controlled by spatiotemporal characteristics. Thus, we demonstrated the same before for the anomalous kinetic isotope effect, neutron control, and mechanochemical conformational transitions  ^{26, 28}.

Changes in the rate of hatching of dormant eggs when irradiated under thermal neutron irradiation may indicate a modulation of redox reactions due to the generation of free radicals. This phenomenon is observed as a result of the decay of unstable isotopes of elements with the release of high-energy photons, followed by ionization of the substance. One of the significant factors influencing the mutual tuning of flickering dipoles, and, accordingly, the intensity of thermal radio emission of particles, is the microelement environment. 

When the elemental composition changes in a particular sample, the flux density of thermal radio emission will undergo changes. This phenomenon is explained by the redistribution of dipoles in supramolecular formations and changes in the dielectric constant of the medium. Since the results obtained by X-ray fluorescence spectroscopy directly depend on the organic microenvironment, monitoring the microelement composition with the proper level of reliability in order to analyze conformational transitions in the complex organic matrix using the XRF method is impossible without making a reference sample due to specific matrix effects affecting the X-ray fluorescence yield ²⁹.

Sample preparation by the technique of preparing a reference sample is accompanied by a change in the organic matrix, which is also observed when A. salina is heated for 9 hours at t = 150 ^oC and, as a consequence, a change in the XRF signal.

It has been shown that various biological objects, such as A. salina, Lycopodium sp., intact Kalanchoe leaves, also emit in the thermal radio band when heated from 25 ^oC to 37 ^oC. The Kalanchoe leaf showed the lowest flux density of thermal radio emission due to absorption. When the test sample was heated above 37 ^oC and further the organic matrix was destroyed, that is, in the temperature denaturation or accelerated aging method, a decrease in the thermal radio signal was observed, as well as a change in the intensities of the elements contained in the samples. This phenomenon allows for the development of technique for quality control of catalysts with an organic matrix for the induction of an active conformation.

This study proposes a rapid method for viability control of powdered dry preparations. Lyophilized powder of Kalanchoe daigermontiana, dry dormant eggs of Artemia salina, and dry powder of Lycopodium sp. spores were proposed as model objects. Live and inactivated preparations were characterized by elemental composition and intracellular water content. Suspensions of virus-like particles (VLPs) to SARS-CoV-2 were proposed as a cell-free reference material. 

It was shown that the listed model inactivated samples lost up to 90% of the density of their intrinsic flux of thermal radio emission in the millimeter band. The vitality of lyophilized therapeutics (from medicinal plants to bacterial mixtures that stabilize the intestinal microflora) can be characterized in a rapid manner.

REFERENCES

1. Astashkina AP, Khudyakova LI, Kolbysheva YV. Microbiological quality control of probiotic products. Procedia Chemistry. 2014 Jan;1;(10):74-9. https://doi.org/10.1016/j.proche.2014.10.014

2. Klein-Junior LC, de Souza MR, Viaene J, Bresolin TMB, de Gasper AL, Henriques AT, Heyden YV. Quality Control of Herbal Medicines: From Traditional Techniques to State-of-the-art Approaches. Planta Med. 2021 Oct;87(12-13):964-988. https://doi.org/10.1055/a-1529-8339 PMid:34412146

3. Momcilovic D, Rasooly A. Detection and analysis of animal materials in food and feed. Journal of Food Protection. 2000 Nov;1;63(11):1602-9. doi: 10.4315/0362-028x-63.11.1602. https://doi.org/10.4315/0362-028X-63.11.1602 PMid:11079709

4. Zuber S, Brüssow H. COVID 19: challenges for virologists in the food industry. Microb Biotechnol. 2020 Nov;13(6):1689-1701. https://doi.org/10.1111/1751-7915.13638 PMid:32700430 PMCid:PMC7404336

5. Xue T, Lu Y, Yang H, Hu X, Zhang K, Ren Y, Wu C, Xia X, Deng R, Wang Y. Isothermal RNA Amplification for the Detection of Viable Pathogenic Bacteria to Estimate the Salmonella Virulence for Causing Enteritis. J Agric Food Chem. 2022 Feb 9;70(5):1670-1678. https://doi.org/10.1021/acs.jafc.1c07182 PMid:35099949

6. Andrews M, Baum J, Gilson PR, Wilson DW. Bottoms up! Malaria parasite invasion the right way around. Trends Parasitol. 2023 Dec;39(12):1004-1013. https://doi.org/10.1016/j.pt.2023.09.010 PMid:37827961

7. Syroeshkin AV, Bakeeva LE, Cherepanov DA. Contraction transitions of F1-F0 ATPase during catalytic turnover. Biochimica Et Biophysica Acta (BBA) - Bioenergetics. 1998 Dec;1;1409(2):59-71. https://doi.org/10.1016/S0005-2728(98)00150-9

8. Grimaldi M, Santoro A, Buonocore M, Crivaro C, Funicello N, Saponetti MS, et al. A new approach to supramolecular structure determination in pharmaceutical preparation of Self-Assembling peptides: A case study of lanreotide Autogel. Pharmaceutics. 2022 Mar;20;14(3):681. https://doi.org/10.3390/pharmaceutics14030681 PMid:35336055 PMCid:PMC8954372

9. Kogo T, Utatsu K, Taharabaru T, Onodera R, Motoyama K, Higashi T. Polyrotaxane-Based Supramolecular Material for Improvement of Pharmaceutical Properties of Protein Drugs. J Pharm Sci. 2022 Jul;111(7):2116-2120. https://doi.org/10.1016/j.xphs.2022.01.018 PMid:35093335

10. Sorokin EV, Tsareva TR, Rudneva IA, Timofeev BI, Lyashko AV, Balanova MA, Artemov EK, Grebennikova TV, Timofeeva TA. [Monoclonal antibodies to hemagglutinin of influenza A/H7N3 virus (Orthomyxoviridae: Alphainfluenzavirus: Influenza A virus)]. Vopr Virusol. 2021 Jul 9;66(3):189-197. Russian. https://doi.org/10.36233/0507-4088-45 PMid:34251156

11. Derjaguin BV, Abrikosova II, Lifshitz EM. Molecular attraction of condensed bodies. Physics-Uspekhi 2015 Sep;30;58(9):906-24. https://doi.org/10.3367/UFNe.0185.201509i.0981

12. Stöhr M, Sadhukhan M, Al-Hamdani YS, Hermann J, Tkatchenko A. Coulomb interactions between dipolar quantum fluctuations in van der Waals bound molecules and materials. Nat Commun. 2021 Jan 8;12(1):137. https://doi.org/10.1038/s41467-020-20473-w PMid:33420079 PMCid:PMC7794295

13. Deryagin BV. Thermodynamics of Free, Foam, and Emulsion Films. Kolloidnyj Zhurnal. 1994;56(1) 133-134.

14. Syroeshkin AV, Petrov GV, Taranov VV, Pleteneva TV, Koldina AM, Gaydashev IA, Kolyabina ES, Galkina DA, Sorokina EV, Uspenskaya EV, Kazimova IV, Morozova MA, Lebedeva VV, Cherepushkin SA, Tarabrina IV, Syroeshkin SA, Tertyshnikov AV, Grebennikova TV. Radiothermal Emission of Nanoparticles with a Complex Shape as a Tool for the Quality Control of Pharmaceuticals Containing Biologically Active Nanoparticles. Pharmaceutics. 2023 Mar 16;15(3):966. https://doi.org/10.3390/pharmaceutics15030966 PMid:36986826 PMCid:PMC10059067

15. Melekhov E.I. On the possible principle of regulation of damage and protective reaction of cells, Journal. Gen. Biology, 1983;44(3):386-397.

16. Nikolaeva YG, Frolov VA, Syroeshkin AV. Effect of low temperatures on diapausing cysts of Artemia salina. Natural and Technical Sciences. 2008;3;21-25.

17. Jönsson KI. Radiation Tolerance in Tardigrades: Current Knowledge and Potential Applications in Medicine. Cancers (Basel). 2019 Sep;9;11(9):1333. https://doi.org/10.3390/cancers11091333 PMid:31505739 PMCid:PMC6770827

18. Gajardo GM, Beardmore JA. The brine shrimp artemia: adapted to critical life conditions. Front Physiol. 2012 Jun;22;3:185. https://doi.org/10.3389/fphys.2012.00185 PMid:22737126 PMCid:PMC3381296

19. Aksenov SI, Goriachev SN, Fateeva MV, Nikitina TN. NMR spin echo study of different strains of Saccharomycodes genus yeasts with varying resistance to lyophilization. 1973 Sep;1;5:729-36.

20. Sellami I, Naceur HB, Kacem A. Study of Cysts Biometry and Hatching Percentage of the Brine Shrimp Artemia salina (Linnaeus, 1758) from the Sebkha of Sidi El Hani (Tunisia) According to Successive Generations. Aquaculture Studies. 2020 Dec;17;21(1):41-6. https://doi.org/10.4194/2618-6381-v21_1_05

21.Giacosa JPR, Morbelli MA, Giudice GE, Gorrer DA. Spore morphology and wall ultrastructure of Lycopodiaceae from northwest Argentina. Review of Palaeobotany and Palynology. 2016 Feb; 1;225:84-94. https://doi.org/10.1016/j.revpalbo.2015.11.009

22. Gaidashev IA, Syroeshkin AV. Development of a reference sample for rapid analysis of an elemental composition of medicinal plant raw materials. Int J Appl Pharm. 2024 Mar;16(2). https://doi.org/10.22159/ijap.2024v16i2.49870.

23. Petrov GV, Taranov VV, Syroeshkin AV. Express method for quality control of products after the fluidized bed aerosol chamber by detecting radio thermal emission of nanoparticles. European Chemical Bulletin. 2023;12;6;3035-3041. https://doi.org/10.48047/ecb/2023.12.6.273 .

24. Goncharuk VV, Lapshin VB, Burdeinaya TN, Pleteneva TV, Chernopyatko AS, Atamanenko ID, et al. Physicochemical properties and biological activity of the water depleted of heavy isotopes. Journal of Water Chemistry and Technology. 2011 Feb;1;33(1):8-13.  https://doi.org/10.3103/S1063455X11010024

25. Nikolaeva YG, Nikolaev GM, Timofeev KN, et. al. The study of the state of water in dormant forms of Artemia salina. Vestnik RUDN. 2007;1;5-9.

26. Syroeshkin AV, Tarabrina IV, Morozova MA, Marukhlenko AV, Zlatskiy I, Makarova MP. Possible Mechanisms of Relations between the Thermal Neutrons Field and Biosphere. The Scientific World Journal. 2020 Aug;11;2020:1-5. https://doi.org/10.1155/2020/2175296 PMid:32848512 PMCid:PMC7439186

27. Petrov GV, Galkina DA, Koldina AM, Grebennikova TV, Eliseeva O, Chernoryzh YYu, et al. Controlling the quality of nanodrugs according to their new Property-Radiothermal emission. Pharmaceutics. 2024 Jan 26;16(2):180. https://doi.org/10.3390/pharmaceutics16020180 PMid:38399241 PMCid:PMC10891502

28. Syroeshkin AV, Pleteneva TV, Uspenskaya EV, Zlatskiy I, Antipova NV, Grebennikova TV, et al. D/H control of chemical kinetics in water solutions under low deuterium concentrations. Chemical Engineering Journal. 2019 Dec;1;377:119827. https://doi.org/10.1016/j.cej.2018.08.213

29. Hosry LE, Sok N, Richa R, Mashtoub LA, Cayot P, Bou-Maroun E. Sample preparation and analytical techniques in the determination of trace elements in food: a review. Foods. 2023 Feb 20;12(4):895. https://doi.org/10.3390/foods12040895 PMid:36832970 PMCid:PMC9956155