ISSN 00125016, Doklady Physical Chemistry, 2014, Vol. 456, Part 2, pp. 86–89. © Pleiades Publishing, Ltd., 2014. Original Russian Text © A.I. Konovalov, E.L. Mal’tseva, I.S. Ryzhkina, L.I. Murtazina, Yu.V. Kiseleva, V.V. Kasparov, N.P. Pal’mina, 2014, published in Doklady Akademii Nauk, 2014, Vol. 456, No. 5, pp. 561–564.

PHYSICAL CHEMISTRY

Formation of Nanoassociates Is a Factor Determining Physicochemical and Biological Properties of Highly Diluted Aqueous Solutions Academician A. I. Konovalova, E. L. Mal’tsevab, I. S. Ryzhkinaa, L. I. Murtazinaa,

Yu. V. Kiselevaa, V. V. Kasparovb, and N. P. Pal’minab Received February 10, 2014

DOI: 10.1134/S0012501614060050

Aqueous solutions of many biologically active compounds (BACs) are known to exhibit bioeffects (detectable responses of biological systems to the action of aqueous BAC solutions) both at common (physiological, therapeutic) solute concentrations (10–2–10–7 mol/L) and in the range of highly diluted solutions (10–10–10–20 mol/L) [1–3]. It should be noted that such solutions are usually prepared by serial dilution from stock solutions of higher concentration; therefore, the specified concentrations are calculated values. Bioeffects at common BAC concentrations are not only beyond question but they are also a constituent of essential principles of life sciences and a basis of phar maceutics. Rather, the existence of such effects for highly diluted aqueous solutions, referred to as ultra lowdose effects, was, and is, doubted since the phe nomenon had no satisfactory physicochemical expla nation [1–4]. Moreover, they were in contradiction with the concept of the nature (state) of solutions and remained as if it were outside the (scientific) law. Indeed, whereas the ratio of the number of solute molecules to the number of water molecules at a con centration of 10–2 mol/L is ≈1 : 103, the number of solute molecules in highly diluted solutions is vanish ingly small. In particular, at a solute concentration of 10–17 mol/L, the solutetowater ratio is ≈1 : 1018. Under these conditions, any bioeffects are seemingly absent. Nevertheless, there are thousands (!) of exam ples of manifestation of bioeffects in response to the action of highly diluted aqueous solutions of BACs, including drugs, on biological systems belonging to different biological organization levels (biomacromole cules–cells–organs–organisms–populations) [1–5].

Here, we mean bioeffects that can be reproducibly quantified, which eliminates subjectivity in recogni tion of their existence. There is a scientific controversy. The reason for this controversy is likely that, in highly diluted solutions, a dissolved BAC, i.e., its sep arate molecules, is not directly a factor determining the manifestation of bioeffects of such solutions; rather, some other processes that occur in solution upon dilution are responsible for such a behavior. According to rather extensive physicochemical studies of the team headed by A.I. Konovalov ([6–8] and ref erences therein) dealing with about 100 compounds of different chemical and biological nature in the con centration range from 10–2 to 10–20 mol/L, an unknown phenomenon is observed when highly diluted solutions are prepared by a standard procedure (successive serial dilutions). This phenomenon con sists in formation of nanosized molecular ensembles involving water molecules, referred to as nanoassoci ates. Nanoassociates are detected by dynamic light scattering (DLS). Their size (effective hydrodynamic diameter D) is as large as 400–500 nm. The authors of these studies have assumed that the formation of nanoassociates is an actual reason for “anomalous” physicochemical properties of highly diluted aqueous solutions and can cause the bioeffects of such solutions. It should be noted that the anomaly of the physicochemical properties of highly diluted aqueous solutions consists in that solution properties observed upon dilution differ from the properties of double distilled water (a solvent). At the same time, at high dilutions, such differences in properties, as well as bioeffects, should be absent for the same reasons. Indeed, what physicochemical property, other than a water property, can be exhibited by a solution if it con tains one solute molecule per billion of billions of water molecules (10–17 mol/L)? The above assumption was based on the fact that there is a correlation between biological and physico chemical properties of solutions and nanoassociate parameters [6–8]. However, the question has arisen

a

Arbuzov Institute of Organic and Physical Chemistry, Kazan Research Center, Russian Academy of Sciences, ul. Akademika Arbuzova 8, Kazan, 420083 Tatarstan, Russia email: [email protected] b Emanual Institute of Biochemical Physics, Russian Academy of Sciences, ul. Kosygina 4, Moscow, 119991 Russia 86

FORMATION OF NANOASSOCIATES

about expanding the scientific base, which would give an opportunity to find additional arguments in favor of this assumption. In this context, an important fact is that the above studies were carried out under both “common” condi tions (on the laboratory bench) and “hypoelectromag netic” conditions. In the latter case, solutions after their preparation by a developed procedure were kept before taking measurements for 24 h not on the labo ratory bench as usually, but rather in a permalloy con tainer protecting the contents from the geomagnetic and lowfrequency external electromagnetic fields. A threelayer permalloy container was used in which the geomagnetic field induction decreased more than 1000fold. As a result, a conclusion has been drawn that the existence of external electromagnetic fields is a man datory condition for the formation of nanoassociates. The experiments have shown that, in the absence of electromagnetic fields (shielding by a permalloy con tainer), nanoassociates are not detected by DLS in solutions at concentrations below some threshold dilution level, as a rule, below concentrations 10–5– 10–8 mol/L; i.e., in highly diluted solutions, the prin ciple “no electromagnetic fields, no nanoassociates” is valid. These results allow us to believe that a permalloy container can serve as a tool to verify whether the for mation of nanoassociates in highly diluted aqueous solutions is necessary for the manifestation of both anomalous physicochemical and biological properties (bioeffects) of such solutions. Once this assumption is valid, then if nanoassociates do not form in solutions under magnetic shielding conditions, these solutions should exhibit neither anomalous physicochemical properties nor biological properties. In this study, the assumption was verified for solu tions of potassium phenozan (PP, potassium β(4 hydroxy3,5ditertbutylphenyl)propionate). This is a known watersoluble synthetic antioxidant of the screened phenol class synthesized at the Institute of Biochemical Physics, RAS. It has been previously shown that PP solutions, including highly diluted solutions, exhibit diverse biological activity in vitro and in vivo [1, 9, 10]. Previously [11], series of PP solutions with different degrees of dilution (concen trations) under common and hypoelectromagnetic conditions were studied by DLS, and their electrical conductivity and surface tension were determined. The results important for further consideration are shown in Fig. 1. Figure 1a shows DLS data. In agreement with the above conclusions, the figure demonstrates that, under common conditions, nanoobjects are detected in the entire range of dilutions (concentrations) of PP solu tions. The nanoassociate size changes nonlinearly and nonmonotonically with a change in the degree of dilution (the significance of these changes in this par ticular case will be seen from the subsequent text). At DOKLADY PHYSICAL CHEMISTRY

Vol. 456

Part 2

87

the same time, under hypoelectromagnetic conditions in solutions with concentrations below threshold val ues (at the level 1 × 10–9–1 × 10–7 mol/L), no nanoob jects (nanoassociates) were detected. It unambiguously follows from Figs. 1b and 1c that, as distinct from the common conditions, under hypo electromagnetic conditions, the physicochemical properties of solutions (electrical conductivity and surface tension) beyond the threshold dilutions (con centrations) coincide with those of water. No anoma lous properties under these conditions are observed in this concentration range. On the basis of the data in Fig. 1a, the principle “no electromagnetic fields–no nanoassociates–no anomalous physicochemical properties of solutions” is logically valid. Thus, the assumption that formation of nanoasso ciates in highly diluted solutions is responsible for their anomalous physicochemical properties was unambig uously verified for PP solutions. To confirm the assumption that nanoassociate for mation in highly diluted solutions under common conditions is also responsible for their biological prop erties, an additional study was carried out. To do this, we studied, in vitro, the effect of PP solutions on natural membranes, synaptosomes iso lated in different seasons (spring, autumn) from the brain of F1(C57xDBA2) mice by sequential centrifu gation in the sucrose gradient as described in [12].These synaptosomes are rather homogeneous membrane structures slightly differing in size [13]. A criterion for the effect of PP solutions on synap tosomes was the change in microviscosity of mem brane lipids, which was evaluated from the change in the rotational correlation time (τс) of a spin probe incorporated into membranes as a function of the degree of dilution (concentration) of PP solutions. A spin probe was 16doxyl stearic acid, widely used for this purpose [14]. The τс time was calculated from the EPR spectra recorded on a Bruker EMX EPR spec trometer in an automatic mode at 293 K. The experiments were carried out by the procedure described below. To an analyte sample containing 200 µL of a membrane suspension, 1.5 µL of the spin probe was introduced, the system was incubated for 20 min (the time it takes for the probe to be built in), then 5 µL of a PP solution of specified concentration was added, and EPR spectra were recorded. Two series of PP solutions prepared under common and hypo electromagnetic conditions were studied. The results presented in Fig. 1d show that, for solu tions prepared under common conditions, i.e., con taining nanoassociates, bioeffects are observed at con centrations 1 × 10–6, 1 × 10–12, and 1 × 10–15 mol/L. In solutions prepared under hypoelectromagnetic condi tions, i.e., not containing nanoassociates, the bioef fect is observed at the concentration 1 × 10–6 mol/L; whereas at the other two concentrations (1 × 10–12 and 1 × 10–15 mol/L), the bioeffect is absent. 2014

88

KONOVALOV et al.

Comparison of the data of biological experiments (Fig. 1d) with the data on the size of nanoobjects (Fig. 1a) and surface tension (Fig. 1c) is very illustra tive. For solutions prepared under common conditions, the bioeffect at a concentration of 1 × 10–6 mol/L cor responds to a maximum on the size scale, and the bio effects at 1 × 10–12 and 1 × 10–15 mol/L correspond to minima on the size scale. The size maximum at 1 × 10–6 mol/L, as well as the corresponding bioeffect, persists for solutions prepared under hypoelectromag netic conditions. The surface tension data are evi dence that, for solutions prepared under common conditions, three bioeffects also correspond to three minima, one of which persists for solutions prepared under hypoelectromagnetic conditions, while the other two minima disappear. On the whole, two conclusions can be drawn from all the data obtained. (1) For solutions prepared under common condi tions, nanoobjects and, hence, presumably bioeffects at the concentration 1 × 10–6 mol/L, on the one hand, and those at 1 × 10–12 and 1 × 10–15 mol/L, on the other hand, are of different nature. It should be emphasized that these effects are observed on opposite sides of the threshold concentration of the solutions. In this paper, we restrict ourselves to the statement of these facts. (2) The second conclusion corresponds to the aims of this study. In highly diluted aqueous solutions (at concentrations below threshold ones) prepared under hypoelectromagnetic conditions where nanoassoci ates do not form, bioeffects are absent; i.e., for potas sium phenozan solutions and the biosystem under consideration, the principle “no electromagnetic field–no nanoassociates–no bioeffects” is valid. Thus, our findings demonstrate that in the case of potassium phenozan solutions, formation of nanoas sociates is a factor determining both the physicochem ical and biological properties of highly diluted aqueous solutions. Further studies will show whether this con clusion is of general character and whether it is valid for other systems or it is one of the components of the phenomenon under consideration.

D, nm (a)

500 400

2

1

300 200 100

No nanoassociates

−18 χ, µS/cm 100

−14

сt

−10

−6

−2

−6

−2

−6

−2

(b)

80 60 40 сt

1 20 2 0

−18 σ, mN/m 72

−14

−10 (c)

2

68 1 64 60 56 сt

[(τс, exp− τс, contr.)/τс, contr.] × 100%

52 −18

−14

14

−10 (d)

10

1

6

ACKNOWLEDGMENTS This work was supported by the Presidium of the RAS (program no. 28) and the Russian Foundation for Basic Research (project no. 13–03–00002).

2 2 −2

−18

−14

−10

−6

−2 logc

REFERENCES

Fig. 1. Concentration dependences of (a) the size of parti cles formed in potassium phenosan solutions, (b) electrical conductivity, (c) surface tension, and (d) microviscosity of deeplying lipid layers of synaptosomes from the mouse brain for diluted potassium phenosan solutions prepared (1) under common conditions and (2) kept in a permalloy container.

1. Burlakova, E.B., Konradov, A.A., and Mal’tseva, E.L., Khim. Fiz., 2003, vol. 22, no. 2, pp. 21–40. 2. Ashmarin, I.P., Karazeeva, E.P., and Lelekova, T.V., Zh. Vses. Khim. Ova im. D.I. Mendeleeva, 1999, vol. 43, no. 5, pp. 21–31. 3. Burlakova, E.B., Zh. Vses. Khim. Ova im. D.I. Men deleeva, 2007, vol. 51, no. 1, pp. 3–12.

DOKLADY PHYSICAL CHEMISTRY

Vol. 456

Part 2

2014

FORMATION OF NANOASSOCIATES 4. Mattison, M.P. and Calabrese, E.J., Hormesis: a Revo lution in Biology, Toxicology and Medicine, 1st ed., New York: Springer, 2009. 5. Shimanovskii, N.L., Epinetov, M.A., and Mel’ni kov, M.Ya., Molekulyarnaya i nanofarmakologiya (Mole cular and Nano Pharmagology), Moscow: Fizmatlit, 2010. 6. Konovalov, A.I. and Ryzhkina, I.S., Izv. Akad. Nauk, Ser. Khim., 2014, no. 1, pp. 1–14. 7. Ryzhkina, I.S., Murtazina, L.I., Sherman, E.D., Pantyukova, M.E., Masagutova, E.M., Pavlova, T.P., Fridland, S.V., and Konovalov, A.I., Dokl. Phys. Chem., 2011, vol. 438, part 1, pp. 98–102. 8. Ryzhkina, I.S., Kiseleva, Yu.V., Murtazina, L.I., Pal’mina, N.P., Belov, V.V., Mal’tseva, E.L., Sher man, E.D., Timosheva, A.P., and Konovalov, A.I., Dokl. Phys. Chem., 2011, vol. 438, part 2, pp. 109–113. 9. Mal’tseva, E.L., Pal’mina, N.P., and Burlakova, E.B., Biol. Membr., 1998, vol. 15, pp. 199–212.

DOKLADY PHYSICAL CHEMISTRY

Vol. 456

Part 2

89

10. Pal’mina, N.P., Chasovskaya, T.E., Belov, V.V., and Mal’tseva, E.L., Dokl. Biophys. Biochem., 2012, vol. 443, pp. 100–104. 11. Ryzhkina, I.S., Kiseleva, Yu.V., Murtazina, L.I., and Konovalov, A.I., Dokl. Phys. Chem., 2012, vol. 446, part 1, pp. 153–157. 12. Whittaker, V.P., Meth. Neurochem., 1972, vol. 2, pp. 1– 52. 13. Fleisher, S. and Packer, L., Methods in Enzymology, Fleisher, S. and Packer, L., Eds., New York: Academic Press, 1974, vol. 31. 14. Spin Labeling: Theory and Applications, Berliner, L.J., Ed., New York: Academic Press, 1976. Translated under the title Metod spinovykh metok: Teoriya i prime nenie, Moscow: Mir, 1979.

Translated by G. Kirakosyan

2014