Human Suspended Animation

Reversible human suspended animation / biostasis without cryonics / cryogenic temperatures

1. Another Road to Full Reversibility? T.Theodorus Ibrahim Long Term Medical Biostasis

2. DEFINITION OF SUSPENDED ANIMATION & BIOSTASIS For the purposes of this presentation these are defined as: “ Any state of full or partial suspension of global biochemical-biological activity in an organism irrespective of reversibility or the method used to achieve such a state ”

3. THE PROBLEM: ARTIFICIAL METHODS FOR SIGNIFICANTLY SLOWING OR STOPPING BIOLOGICAL PROCESSES (e.g. HUMAN CRYOPRESERVATION) CAUSE BIOLOGICAL DAMAGE METHOD DAMAGE And a major consequence of this damage is that the state of suspended life thus created is of poor quality and hard to reverse BIOCHEMISTRY Method creates biostasis ….. But damage also created by method….. BIOSTASIS NO RETURN!

4. Chilling Injury! Solute Injury! Ice Mechanical Injury! Cold Shock! TOO FAST! Cryogenic temperatures for example are very effective at stopping biochemical activity but can create damage by temperature drop AND ICE FORMATION….

5. …and attempting to prevent or mitigate this damage using cryoprotectants creates other damage (in many specific and non-specific ways) Etc!

6. SO HOW CAN WE CREATE NON-DAMAGING (AND THUS MORE REVERSIBLE) FORMS OF SUSPENDED ANIMATION?

7. PHYSICS CHEMISTRY BIOLOGY BY USING MANY BIOSTASIS INDUCING METHODS FROM MULTIPLE DOMAINS (i.e. Physics, Chemistry, Biology) TOGETHER METHOD 3 METHOD 2 METHOD 6 METHOD 5 METHOD 9 METHOD 8 METHOD 1 METHOD 4 METHOD 7

8. METHOD 2 WHY USE MULTIPLE METHODS & DOMAINS? Because enough methods may ADD up to create sufficient biochemical slowdown for suspended animation but the damage each creates might NOT add together in an equivalent way METHOD 1 METHOD 2 THRESHOLD Threshold for serious damage not reached Threshold for biostasis reached & passed DAMAGE 1 DAMAGE 2 DAMAGE 2 DAMAGE 1 METHOD 1

9. Analogy : standing or lying on a bed of nails is not harmful if there are enough of them! WHY MIGHT THE DAMAGE NOT ADD TOGETHER? Because having enough methods and making them different enough allows the body to ‘compartmentalise’, spread and dilute their damage and so be better able to deal with it

10. Therefore as each new method is added, it contributes to biological slowdown but the damaging effects are never able to accumulate to the same extent

11. In addition the very capacity for damage to occur is reduced at source by applying the following FIVE DAMAGE REDUCTION RULES

12. CHEMICAL PHYSICAL BIOLOGICAL Polymer Sterics Bonding WHAT ARE THE MULTIPLE METHODS FOR CREATING BIOSTASIS? SIX ARE PROPOSED (Initially....) Anhydrousity Laser Chemo Interdiction Low Temperature Metabolic Depression

13. HOW DO THE METHODS ‘ADD UP’? Each method contributes SYNERGISTICALLY to biochemical slowdown in a semi-exponential and non-linear way. There is far more slowdown than we might expect as each new method is added SLOWDOWN

14. DETAILS ABOUT THE METHODS DETAILS ABOUT THE METHODS

15. PHYSICAL Temperature LOW TEMPERATURE (-3 °C / 270 K ) SUPERCOOLING only (done fast). No use or minimal use of cryoprotectants of types and at concentrations shown to be non-toxic (to all cell types) supplemented by additional protective measures against toxicity and ice formation Simple blood infusion / circuit. Viaspan base with: 3-O-methyl-D-glucose Trehalose Hydrogel monomers Anti freeze protein PEG–35kD (5%) Magnetocaloric-NIR device vesicles We rely on ultra rapid cooling (>3°C per minute) as the chief protective mechanism against ischemia during the descent phase from normothermic temperatures and this is achieved by combining four principal methods: 1. Polyatomic laser cooling 2. Perfluorocarbon lung lavage 3. Body glove graphene / chilled liquid cooling 4. Magnetocaloric NIR cooling Wan et al (2018) have already demonstrated 1 hour storage of rat hearts at - 4°C (under 41 MPa pressure) without using any cryoprotectant at all and with excellent post storage recovery of function. Hence this is the foundation strategy and extended to the whole body Non absorbable endpoint Magnetocaloric-NIR Radiation Pressure [41 MPa] Temperature [-3°C / 270 K] Non absorbable Endpoint Laser Cooling Radiation Body glove made from vascularised graphene fibre & using graphene particulate chilled liquid as coolant 10 Tesla / 100 Hz magnetic field generator for magnetocaloric-NIR device vesicles Lung Lavage Tube Body Glove Liquid Exit Tube Body Glove Liquid Entry Tube Laser Cooling Device

16. BIOLOGICAL Metabolic Depression Transcription inhibition Oxidative Phosphorylation Shutdown Enzyme Shutdown ATP Protein synthesis shut down Hibernating animal gene activation pattern (e.g. bear) Pharmacological Agents (Gene product copies) Hydrogen Sulfide 5’-Adenosine Monophosphate Gene Knock-down Agents METABOLIC DEPRESSION Replicated (including protective processes) via The biology of natural hibernation is copied into humans & additional factors applied THIS RESULTS IN Inert Non-Metabolizable bio-analogous end product exert negative feedback / competitive inhibition on all major metabolic pathways Small Molecules (examples) Kinetics and

17. Other Hibernating Animal Orthologous Gene Activation Upregulation AND Downregulation Patterns to Copy Include:

18. But….which orthologous genes do we copy? Just copy them. COPY THEM ALL

19. LASER / EM FIELD CHEMOINTERDICTION PHYSICAL Laser / EM Field Chemo Interdiction 4. Protein Functional Resonance (Cosic & non-Cosic) LASER / ELECTROMAGNETIC RADIATION CAN CONTROL CHEMICAL & BIOCHEMICAL REACTIONS: IT CAN SELECT THEIR PATHWAYS, CAUSE THEM TO SPEED UP, SLOW DOWN, OR EFFECTIVELY STOP 2. ‘No Go’ Selective Excitation of Reagent Vibrational Modes 3. Control of Reagent Approach Geometry 1. Control of Orbital Alignment 5. Increase Required Activation Energy / Barrier SOME EXAMPLE CHEMOINTERDICITON METHODS

20. Each chemical process is targeted for slowdown using a specifically tailored deep penetrating infrared laser field (≈800nm-1500nm) which incorporates the different interdiction methods . . . ≈ 5000 Chemical reaction interdiction methods Infrared radiation incorporating OAM channels OAM Channels Every tailored IR laser field has its own optical angular momentum (OAM) channel (total ≈5000) which are multiplexed together thus allowing simultaneous targeting of all metabolic pathways

21. PHYSICAL POLYMERSTERICS Biocompatible polymers (including hydrogels) self assemble in vivo and physically block / sterically hinder / ‘cage’ molecular biological / biochemical activity thus stopping it. It is a form of semi-‘plastination’ / whole body ‘gentle’ histological tissue embedment Polymer Sterics Made externally & perfused and / or made internally via DNA – RNA - protein synthesis Monomers Polymerisation OR Depolymerisation is activated when appropriate by penetrating laser light of the correct frequency or by a trigger temperature Proteins can’t move Polymer Thread Laser Light Temperature Polymer Mesh Monomers can be BOTH simple molecular or supramolecular e.g. Poly-(R)-3-hydroxybutyrate Poly(trimethylene carbonate) (passive diffusion is too slow)

22. Schematic illustrations of the on/off switching of F1-ATPase rotation by entanglement with supramolecular hydrogel fibre nanomesh [Ye et al 2014)  ALL molecular machinery can be switched off (when monomers are polymerised) and then switched back on (when polymers are depolymerised)

23. Van der Waals CHEMICAL Bonding CHEMICAL BONDING Polypeptide aptamers make multiple types of biologically benign reversible bonds between macromolecules to create pervasive, rigid crosslinking throughout cells and tissues Cation- Π Interaction 1 Hydrogen Bond 2 Sulfide Bridge 3 Ionic/ Electrostatic 4 The six types of bonds aptamers can make: five specific ones and additional Van der Waals interactions Π - Π Interaction 6 5 5 4 6 1 5 2 3 2 Example Aptamer [NB: Parts not to scale]

24. Each aptamer binds to two or more macromolecules locking them together and each macromolecule is also able to form further attachments with other aptamers thus creating a crosslinked network of molecules Molecule1 Molecule2 Aptamer Molecules Bound to Aptamer Example Section of Crosslinked Network [NB: Not to scale]

25. Binding strength of aptamer crosslinking can approach that seen generated via ‘normal’ glutaraldehyde or other histological fixative. Whilst strength of individual bond types (e.g. hydrogen bonds) in aptamer binding is smaller, their greater number creates a similar or greater aggregate effect. In addition, the sulfide bridge bond utilised by aptamers is fully covalent All proposed bonds (e.g. di-sulfide bridges) are biologically ‘familiar’ to the organism (unlike those made by glutaraldehyde) and therefore benign Molecular ultrastructure order is maintained. One of the mechanisms of toxicity by fixatives such as glutaraldehyde is the spatial disorder they cause by forcing misalignment of molecules or parts of molecules they bind to thru ‘pulling’ them away from their normal axial orientation or placement with respect to neighbouring molecular components. Aptamer binding however maintains those spatial orientations SOME KEY FEATURES OF APTAMER BASED CROSSLINKING Protein conformation is conserved. Reacting centres on aptamers are spatially organised so binding only occurs on non-critical places on the target endogenous molecules (e.g. nonessential thiol (SH) groups etc). Intra-protein binding is also avoided. There is therefore no perturbation of protein secondary / tertiary conformation e.g. of alpha helixes

26. The second (more minor) form of chemical bonding is via ultra-dilute solutions of the least toxic traditional fixatives selected for cleavability / vulnerability to non-toxic reversal agents & applied at times where they can do the least damage (principally low temperatures) and at concentrations shown to be recoverable by biological systems Initial possibilities are: Some reversal agents might include:

27. PHYSICAL Anhydrousity ANHYDROUSITY Removing the ‘background’ medium (in this case, water) which enables molecular activity stops such activity from happening H 2 O Aquaporins expel water Protective Trehalose + Entry Agent Hydrogel captures water Protective Intrinsically Disordered Proteins + Entry Agent High solute concentration IN Diluted solute concentration OUT H 2 O REMOVED (but not hydration shell bound water) Osmotic Gradient High water concentration and Low Solutes Low water concentration and High Solutes Intracellular Production

28. EACH METHOD IS APPLIED IN SEQUENCE ( THOUGH WITH SOME OVERLAP ) The sequence is broadly determined by: What is possible in practice e.g. co-ordinated metabolic depression requires a mostly intact biochemistry and hence this must be applied before the high curtailment of molecular motion which results from the application of (for example) polymersteric hinderance Any method able to offer protection against damage caused by another method is placed before that one in the sequence and hence is in ready to act before such damage can manifest 1 3 4 5 6 2

29. HOW MUCH SLOWDOWN CAN WE EXPECT? Metabolic slowdown is calculated on the basis of the Q10 Rule i.e. a 50% drop in metabolic rate for every 10°C reduction of temperature. This predicts a rate of approximately 6.25% of normal metabolic rate at -3°C / 270 K (from four halving's of rate) Each of the other methods is (conservatively) assumed to be capable of at least this amount of slowdown. When applied in sequence, each method compounds with previous ones synergistically creating an increasing level of metabolic rate reduction which finally reaches 0.00000003% of normal metabolic rate when all methods have been applied (last column in chart) All methods applied No methods applied

30. Metabolic slowdown can be represented in terms of how much biological time passes for each 100 years of real time. Using five methods (Low Temp + Biological Metabolic Depression + Laser Chemo Interdiction + Chemical Bonding + Polymersteric Hindrance) results in 1.56 minutes passing for every 100 years of normal time. This is within tolerable limits of ischemic injury (though given we are employing active measures for metabolic depression the actual level of ischemic injury will be a small fraction of this) and hence this points to the possibility of multi century biostasis. The addition of the final method, Anhydrousity, results in a slowdown to 0.02 minutes (1.2 seconds) for every 100 years of real time. This more strongly suggests the possibility of multi-century (or longer) medical biostasis. 52,416,000 minutes equals 100 years (this is the baseline when nothing is used) This is 1.2 seconds

31. CONCLUSIONS

32. Questions, Comments & Complaints [email protected] https://www.facebook.com/t.theodorus.ibrahim An individual of the hibernating species Urocitellus parryii (arctic ground squirrel) teaching us how it’s done