Oral report on xe in dresden

Possible Mechanisms Possible Mechanisms of Cryoprotective Effect of Xenonof Cryoprotective Effect of Xenon

Igor V. Artyuhov (presenting author)Igor V. Artyuhov (presenting author) Alexander Yu. PulverAlexander Yu. Pulver Alex G. PeregudovAlex G. Peregudov Vassilii I. ArtyukhovVassilii I. Artyukhov

Contact: [email protected]: [email protected]

Applied Cryobiology – Scientific Symposium on CryonicsApplied Cryobiology – Scientific Symposium on Cryonics

October 4-5th, Dresden, GermanyOctober 4-5th, Dresden, Germany

AbstractAbstractUse of xenon (and, possibly, other inert gases) for cryopreservation has been proposed as early as in

1960s. Our recent experiments (discussed in another report at this conference*) show a certain promise of this approach as well. Nevertheless, mechanisms of xenon action are still poorly understood. There can be several possible (mutually non-exclusive) explanations:

1. Accumulation of hydrophobic Xe atoms in the cellular lipid membranes providing membranoprotective effect, or in hydrophobic "pockets" of proteins, thus stabilizing them and preventing them from denaturation.

2. Formation of crystals of Xe clathrate hydrate rather than that of ice, possibly with finer grain size or less damaging shape (ice crystals are needle-like, while Xe hydrate crystals are grain-like).

3. Accumulation of Xe atoms in the interfacial layer between liquid water and ice slowing the growth of the latter (ice-blocking effect).

4. Vitrification of Xe water solution.

In the study** we focus on the first three items from this list. We have conducted a large scale molecular dynamics modeling of processes in aqueous Xe solution near and below freezing point. Formation of Xe crystallohydrates and the effect of Xe on the growth of ice crystals are investigated. Some promising implications of our study results for cryoprotection are discussed.

____________________________* A. Pulver, A. Tselikovsky, N. Pulver, I. Artyuhov, V. I. Artyukhov, A. Peregudov. Application of clathrate forming gases for vitrification of organs and whole organisms.** V. I. Artyukhov, A. Yu. Pulver, A. Peregudov, I. Artyuhov. Can xenon in water inhibit ice growth? Molecular dynamics of phase transitions in water-Xe system. J. Chem. Phys. 141, 034503 (2014), doi: 10.1063/1.4887069

Robert W. Prehoda, Suspended animation: the research possibility that may allow man to conquer the limiting chains of time, Chilton Book Co., 1969.

Patent by P. Shcherbakov and V. TelpuhovPatent by P. Shcherbakov and V. Telpuhov

«Biological object (heart, kidney, etc) should be cooled in water up to 0° C at simultaneous saturation with the mixture of xenon, krypton, argon at the ratio of 2.5:47.5:50 rot.%, then one should force out water with gaseous mixture mentioned and at the pressure of 1.5 atm. it is necessary to decrease temperature up to -43° C, then one should decrease the pressure of gaseous medium up to normal level and continue cooling up to -196° C. The innovation enables to achieve reliable and prolonged cryoconservation of animal heart being the constituent of the whole organism (in situ), in a transferred transplant conserved for 6 h against the time of cold cardioplegia and up to the moment of restoring contractile activity. Moreover, it has been possible to achieve restoration of adequate cardiac activity.»

Shcherbakov P.V., Telpuhov V.I. Method for organs and tissues cryoconservation in situ. http://russianpatents.com/patent/226/2268590.html

Physiological properties of xenonPhysiological properties of xenon

• Absolutely non-toxic;• Rapidly both penetrates tissues and leaves

them;• Perfect anaesthetic?;• Suppresses cellular metabolism?;• Acts as antihypoxant?;• Reduces inflammation?.___________________? Nature of these abilities is not known yet…

Issues with common cryoprotectantsIssues with common cryoprotectants• Need for enormous concentrations that inevitably are

toxic;

• Need for very high cooling rates in order to achieve vitrification;

• Problems with perfusion because of high viscosity at low temperatures and high concentrations;

• Poor penetration both when entering the cell and when leaving it;

• Need for very high warming rates in order to avoid crystallisation;

• Problems with non-uniform washout from organ/organism.

Three questionsThree questions

1. Does xenon really act as a cryoprotectant?

2. If 'Yes', then what are the mechanisms?

3. How can it be applied in practice, especially for cryopreservation of bulk biological objects?

Possible (mutually non-exclusive) Possible (mutually non-exclusive) explanationsexplanations

1.Accumulation in cell membranes preventing liquid crystal to gel phase transition and thus maintaining membrane elasticity, or/and in protein molecules' hydrophobic "pockets" preventing denaturation.

2.Formation of xenon hydrate crystals that compete with ice for water molecules, and are for some reason less destructive for cells and tissues. For example they can be finer and/or have less damaging shape, e.g. granular instead of needle-like that of ice.

3.Ice-blocking effect due to accumulation in the ice-water interface zone.

4.Vitrification of xenon-water solution. Its conditions may differ from that of pristine water.

Xenon in cellular membranesXenon in cellular membranes

From: R.D. Booker, A.K. Sum. Biophysical changes induced by xenon on phospholipid bilayers. Biochimica et Biophysica Acta 1828 (2013) 1347–1356


1.Accumulation of xenon in cell membranes, preventing liquid crystal to gel phase transition and thus maintaining membrane elasticity or/and in protein molecules' hydrophobic "pockets" thus preventing them from denaturation. - probable!

2.Formation of xenon hydrate crystals which compete with ice ones for water molecules and are for some reason less destructive for cells and tissues. For example they can be finer and/or have less damaging shape, e.g. granular instead of needle-like that of ice.

3. Ice-blocking effect of xenon due to it's accumulation in the ice-water interface zone.

4.Vitrification of xenon-water solution. It's conditions may differ from that of pristine water.

Vasilii I. Artyukhov et al., Can xenon in water inhibit ice growth? Molecular dynamics of phase transitions in water–Xe system. JCP 141, 034503 (2014)

Some computational detailsSome computational details

Modelling software: GROMACS*

Time step 2 fs

Number of water molecules 4000

Number of xenon atoms 40, 80, 120, 160, 200

* D. van der Spoel et al, J. Comput. Chem. 26, 1701–1718 (2005).

Solubility of xenon in waterSolubility of xenon in water

T,°C

Solubility

-25 -20 -15 -10 -5 5 10 15 20 25 30 35 40 45 50 55 60 65 70 75

0.001

0.002

0.003

0.004

0.005

0.006

0.007

0.008

0.009

0.01

0

Extrapolation

Semi-empirical dependence ln s = A + B/T + C ln T

T,°K 280 270 265 260 255 250

1% 80 80 50 100 100 >100

2% 50 50 50 100 100 >100

3% 30 30 50 100 100 >100

4% 30 30 100 100 100 >100

5% 30 50 100 100 100 >100

*Homogenous crystallization observed

Temperature/Concentration Temperature/Concentration combinations studied: combinations studied:

Duration of simulation runs (ns)Duration of simulation runs (ns)

Radial Distribution Function Xe-O at 260°KRadial Distribution Function Xe-O at 260°K

Solvation shell around Xe atom in waterSolvation shell around Xe atom in water

Source: D. Paschek, J. Chem. Phys. 120, 6674 (2004).

Crystallohydrate of xenonCrystallohydrate of xenon

Homogenous nucleation Homogenous nucleation at the 5% mol. Xe/mol. H2O system at 270°Kat the 5% mol. Xe/mol. H2O system at 270°K

Two microcrystals of xenon clathrate hydrateTwo microcrystals of xenon clathrate hydrate



2.Formation of xenon hydrate crystals which compete with ice ones for water molecules and are for some reason less destructive for cells and tissues. For example they can be finer and/or have less damaging shape, e.g. granular instead of needle-like that of ice. - probable!

3.Ice-blocking effect of xenon due to it's accumulation in the ice-water interface zone.


Systems with iceSystems with ice

(a) pure water at 275°K, (b) 1% mol. Xe/mol. H2O solution at 274°K, (c) 4% mol. Xe/mol. H2O solution at 271°K. (d) Structure of ice–liquid interface: Xe and O atom density profiles for

systems (a) and (c).




3.Ice-blocking effect of xenon due to it's accumulation in the ice-water interface zone. - unlikely...


Dependence of Xe diffusion constant Dependence of Xe diffusion constant on concentration and temperatureon concentration and temperature

Extrapolation of Diffusion constant Extrapolation of Diffusion constant to lower temperaturesto lower temperatures

245 250 255 260 265 270 275 280 2850

0.5

1

1.5

2

D, H20 D, 1% Xe D extrapol.

T0 extrapol= 248.7°K; Is it Tg?!




3.Ice-blocking effect of xenon due to it's accumulation in the ice-water interface zone. - unlikely...

4.Vitrification of xenon-water solution. It's conditions may differ from that of pristine water. - Could be? At ~-25°C?!

Take-home messageTake-home message

• Xenon may serve as a cryoprotectant coming with several valuable features at once – and

no drawbacks at all.

• Most intriguing: possible (not yet established) ability to vitrify water as high as -10’s °C

• Can be used in combination with tolerable concentrations of common cryoprotectors.

• We are open to any form of collaboration in further investigation of this possibility.

Some referencesSome references1. R. W. Prehoda, Suspended animation: the research possibility that may allow man to conquer the limiting chains of time,

Chilton Book Co., 1969

2. S. Sheleg et al., Cardiac Mitochondrial Membrane Stability after Deep Hypothermia using a Xenon Clathrate Cryostasis Protocol – an Electron Microscopy Study. Int J Clin Exp Pathol (2008) 1, 440-447

3. D. S. Laptev et al., The Use of Inert Gas Xenon for Cryopreservation of Leukocytes. Bulletin of Experimental Biology and Medicine, Vol. 157, No. 2, June, 2014, pp. 282-284

4. Shcherbakov P.V., Telpuhov V.I. Gas Influenced Immortality. Chemistry and Life 2006;8:34-39 (in Russian).

5. Shcherbakov P.V., Telpuhov V.I. Method for organs and tissues cryoconservation in situ. http://russianpatents.com/patent/226/2268590.html, http://www.findpatent.ru/patent/226/2268590.html (in Russian)

6. Vasilii I. Artyukhov et al., Can xenon in water inhibit ice growth? Molecular dynamics of phase transitions in water–Xe system. JCP 141, 034503 (2014)

7. R.D. Booker, A.K. Sum. Biophysical changes induced by xenon on phospholipid bilayers. Biochimica et Biophysica Acta 1828 (2013) 1347–1356

8. Rodin V.V., Isangalin F.Sh., Volkov V.Ya. Structure of water protein solutions in a presence of xenon clathrate. Cryobiology & Cryo-Medicine. Kiev: Naukova Dumka, 1984, pp. 3-7 (in Russian).

9. Rodin V.V., Novikov I.A., Volkov V.Ya. Investigation of the influence of xenon on the DNA-bound water system using the method of NMR. Cryobiology 1989;4:35-38 (in Russian)

10. Ryan D. Booker et al., Xenon Hydrate Dissociation Measurements With Model Protein Systems. J. Phys. Chem. B 2011, 115, 10270–10276

Oral report on xe in dresden

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