The Role of Natural Deep Eutectic Solvents - ACS Publications

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How do animals survive extreme temperature amplitudes? The role of Natural Deep Eutectic Solvents Ana Catarina Gertrudes, Rita Craveiro, Zahara Eltayari, Rui L. Reis, Alexandre Paiva, and Ana Rita Cruz Duarte ACS Sustainable Chem. Eng., Just Accepted Manuscript • DOI: 10.1021/ acssuschemeng.7b01707 • Publication Date (Web): 20 Sep 2017 Downloaded from http://pubs.acs.org on September 21, 2017

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How do animals survive extreme temperature amplitudes? The role of Natural Deep Eutectic Solvents Ana Gertrudes 1,2, Rita Craveiro,3 Zahara Eltayari3, Rui L. Reis1,2, Alexandre Paiva3, Ana Rita C. Duarte 1,2*

1

3B’s Research Group- Biomaterials, Biodegradable and Biomimetic, University of Minho,

Headquarters of the European Institute of Excellence on Tissue Engineering and Regenerative Medicine, Avepark 4805-017 Barco, Guimarães, Portugal 2 3

ICVS/3B’s PT Government Associated Laboratory, Braga/Guimarães, Portugal LAQV-REQUIMTE, Departamento de Química, Faculdade de Ciências e Tecnologia,

Universidade Nova de Lisboa, 2829-516 Caparica, Portugal *Correspondence to: [email protected].

Abstract: Recent findings have reported the reason why some living beings are able to withstand the huge thermal amplitudes between winter and summer in their natural habitats. They are able to produce metabolites decreasing deeply the crystallization temperature of water, avoiding cell disrupture due to the presence of ice crystals and overcoming osmotic effects. In vitro, the possibility to cool living cells and tissues to cryogenic temperatures in the absence of ice can be achieved through a vitrification process. Vitrification has been suggested as an alternative approach to cryopreservation and could hereafter follow an interesting biomimetic perspective. The metabolites produced by these animals are mostly sugars, organic acids, choline derivatives or urea. When combined at a particular composition these compounds form a new liquid phase which has been defined as Natural Deep Eutectic Solvents (NADES). In this review we relate the findings of different areas of knowledge from evolutive biology, cryobiology and thermodynamics and give a perspective to the potential of NADES in the development of new cryoprotective agents.

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One Sentence Summary: NADES - the key to survive extreme temperature amplitudes - A biomimetic approach towards the development of new cryoprotectants. Keywords: Deep eutectic solvents; cryopreservation; vitrification; cryoprotective agents; glycerol

Introduction The ability of living beings to adapt to extreme environments has intrigued and passionate many scientists from different fields of research. Likewise the ability to survive extreme temperature amplitudes throughout the year is also remarkable. The understanding of the capacity of living beings to adapt to these conditions may reveal important cues for the development of efficient methods for organ cryopreservation. Nature has provided solutions to allow living beings to inhabit locations with extreme temperature differences and these living beings have adapted themselves to be able to overcome the potential chilling injury in the tissues. In this paper we review different animal species which have been studied and their mechanisms of survival. Figure 1 illustrates the geographic distribution of the animal species which have been studied and which will be here reviewed.

Fig. 1. Geographical distribution of the animal species reviewed.

Mechanisms to survive extreme temperature amplitudes

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Considering for example Alaska, the temperature differences between winter and summer may reach more than 60 ºC 1. Cold temperature triggers chilling injury of the living systems, mainly by ice nucleation and osmotic stress. The decrease in temperature promotes a new equilibrium where available water in the liquid state tends to form ice crystals

2,3

. These can disrupt the

biological structures, namely cell membranes, organelles and extracellular matrix in tissues and organs 4. For this reason, the size and localization of ice crystals formed during cryopreservation has shown to be determinant for survival rates 3. Furthermore, the reduction of available liquid water increases the concentration of the solutes in the cells, inducing osmotic stress

4,5

. The

strategy that living organism have found to survive extreme temperature amplitudes relies on the orchestrated production of different biological metabolites. For example, in the case of stonefly from Alaska (Nemoura arctica), glycerol has been found to increase by three orders of magnitude in cold acclimation, leading to a survival rate of 85% when they are subjected to -15 ºC for 2.5 weeks 6. A group of exceptional animals, with a size of microns, is tardigades. Tardigades, can survive in extreme conditions, hot and cold temperatures, high saline concentrations and ionic radiation 7,8. These resistant small invertebrates support a dehydrated state in cold conditions for long periods, and later can be revived by rehydration. In the 90’s Westh and Ramlov study the variation of trehalose in tardigrades when subjected to dehydration. Trehalose quantity increases near 23 fold during dehydration, and the level is reduced when hydration is initiated

9,10

. Later, they related

the trehalose level and the desiccant state with the increased freeze tolerance

11

. Not only the

metabolites accumulated by the organisms are crucial but also the amount of water that remains in the system during the freezing. In 1995, Somme and Meier reported the survival of hydrated tardigrades after almost 600 days at -20 ºC, and dehydrated tardigrades for 3040 days at the same temperature 7. Furthermore, trehalose was found to be accumulated in several other animals with cold resistance, namely beetles, cabage root fly, gall fly, stonefly, and damaging pests as codling moth and rice weevil 6,12–19 Beetles from central Europe (Ips typographus) endure winter temperatures of -20 ºC, and it has been confirmed that seasonal production of trehalose, other sugars and polyols is part of a strategy for cold survival. During autumn, accumulation of trehalose and glucose was observed, and then, when conditions undergo a threshold point (humidity and temperature), a massive production of polyols was initiated

12

. In spring the identified cryoprotective metabolites were

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practically cleared from the tissues

20

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. Also it has been noticed that available free water in the

organism follows an inverse pattern of lowering during cold to be again increased in the spring 20,21

. Sformo and co-workers not only demonstrated that Alaska beetle larvae can survive below

subzero temperatures but also that vitrification of water occurs in the tissues. Differential scanning calorimetry (DSC) was used to identify the glass transition temperature (Tg) of beetle larvae sealed in the aluminum pan, cooled from 10 ºC to -150 ºC and then warmed-up, applying different rates

21

. Interesting to notice is the fact that two glass transition temperatures were

identified suggesting two different compartments, one richer in water than the other. In a vitrification process the ice nucleation is inhibited

5,22

and this detail is the reason why

vitrification is considered an “ice-free” cryopreservation technique. In the case of earthworms, DSC analysis has shown no evidence of ice formation up to -60 ºC

23

. In the winter season

earthworms specimen can inhabit soils with low subzero temperatures

24

, where glycogen

catabolism and amino acid content has been shown to occur is response to body fluid freezing 25,26

. A two step approach to survive lethal cold environments seems to be used by these animals:

first a slow absorption of sugar to prepare the tissues for the significant osmolality increment then, in a second step, the polyols are promptly produced when the cold hits 27. Polyols found in cold resistant living beings comprehends for example sorbitol, mannitol, erythritol, threitol, arabinitol and ribitol 6,12,16. Similar dual strategy was been observed in other insects, such as Rice Water Weevil, Coldig moth and many others 16,17,19,28. This overwinter tactic seems to work as a progressive creation of a system comprising sugars which are mixed with polyols. Moreover, the observation of amphibians, led to discover another two step approach for cold resistance in wood frogs. For a long time that various specimens of frogs are known to survive subzero conditions. Glucose was the first metabolite to be detected in cold resistance strategy of frogs

29,30

, and its synthesis

regulation was found to be modified in frogs that can tolerate freezing 31,32. For example, higher amounts of membrane glucose transporters are expressed in cold tolerant frogs than in intolerant specimens

33

. Most recently urea was also found to be part of the cold resistance strategy of

terrestrial frogs. Urea is a well-known nitrogenous waste that can denaturate proteins and be highly toxic. However, urea action in the biological systems can be modified by particular solvent conditions

34,35

. Marine beings were found to accumulate not only urea but also betaine,

trimethylamine oxide (TMAO) and sarcosine in stressful biochemical situations 27. And after has

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been proven that addition of urea and betaine in specific quantities allows mammalian cells to survive in high saline media conditions

34

. Moreover, recent studies show that unique solvent

systems comprising urea can even lead to protein stabilization 35. The wood frog Rana sylvatica initiates a process of urea accumulation when drought occurs in its habitat, normally in autumn and early winter. Costanzo and Lee work, registered a 25 fold higher urea content in outdoor individuals collected in November compared to the ones collected in April 36. When temperature drops to near -20 ºC, an increased in glucose production is triggered 36,37. Very similar to insects, the two step strategy starts with urea accumulation, and then the cold freeze initiates the glucose increment, balancing osmolality of the tissues to avoid osmotic stress during water freezing. These natural cryoprotectants flow into the frog’s cells and serve to diffuse the concentration gradient between the interior and exterior of the cells as extracellular water freezes, preventing cell shrinkage. Comparative analysis of organs from frozen and unfrozen test frogs show, not only that glucose was increased in the frozen subject, but the water content in the tissue was reduced 36. Moreover, the levels of urea and glucose accumulated for cold resistance strategy in frogs have been confirmed to be an adaption of a population to the habitat characteristics. In 2013, Costanzo and co-works demonstrated that wood frog populations from Alaska, had higher levels of protective metabolites and had better survival rates in extreme conditions than the ones from Ohio 31. Freeze-tolerant terrestrial frogs survival approach also appear to work as a gradual construction of a system with urea mixed with sugars, in an analogous process to insects strategy. Figure 2 presents a schematic representation of this dual step strategy for overwinter survival. In both systems other metabolites can remain to unraveled, just recently amino acids were discovered to be part of wood frogs preparation for winter cold 38.

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Fig. 2. Schematic representation of dual step strategy for overwinter survival in cold-tolerant insects and frogs

A summary of the different biological metabolites which are synthesized by the different organisms to ensure that life is possible below 0 ºC, is listed in table 1. Besides the presented natural cryoprotectants, many other low weight molecules are produced during drought and cold conditions. For instance, the variation of free amino acids concentration in animals during cold has been described in some animals. Proline content has slightly increased when comparing cold and warm acclimated stoneflies

6

while other aminoacids, such as

glutamine, alanine, lysine, leucine and phenylalanine have been reported in insects, earthworms and amphibians associated to sugars and polyols as part of the cryoprotective strategy 14,17,26,38. This ability of some animal to vitrify or to reduce the crystallization temperature of water inside its cells for winter survival provides insights to answer unmet needs of cryopreservation for tissues and organs.

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Table 1. Metabolites found to be responsible for the overwinter survival of animals living in environments with extreme temperature amplitudes.

Animal

Component 1

Component 2

Component 3

Component 4

Ref

Tardigrades

Trehalose

-

-

-

9,11

Beetles

Trehalose

Glucose

Polyols (sorbitol and

-

12

mannitol) Beetles

Glycerol

-

-

-

21

Cabbage root

Trehalose

-

-

-

13

Trehalose

Glucose

Amino acids

-

14

fly Common fruit fly

(glycine, lysine and tryptophan)

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Stonefly

Trehalose

Glycerol

Proline

-

6

Codling moth

Trehalose

Other sugars

Polyols (manitol and

Amino acids

16

(frutose and

sorbitol)

(alanine)

Trehalose

-

15,18

-

-

19

-

-

26

-

-

14

glucose) Gall fly

Glycerol

Polyols (sorbitol)

Rice weevil

Trehalose

Polyols (sorbitol and mannitol)

Earthworm

Glucose

Amino acids (proline, glutamine and alanine)

Earthworm

Glucose

Frog

Urea

Glucose

-

-

31,36

Frog

Urea

Glucose

Amino acids

-

38

(aspartic acid, alanine, glutamatic acid, leucine, isoleucine, valine, aspargine, serine)

Cryopreservation and Vitrification Cryopreservation is a method to store biological systems for a significant period of time and then recover them with no changes in the metabolism and little or no damage to the biological structure. Cryopreservation combines extreme cold temperatures, to ensure months or even years of storing, with cryoprotectant agents, used to protect cells during freezing and thawing steps 4. Vast development was achieved in the XXth century regarding preservation of cells, tissues and organs with relevant impact in different aspects of our society 39. Simple and robust methods for

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single cell cryopreservation made possible the generalization of in vitro cellular studies, with high contribution for biological and medical scientific developments worldwide. Commercially, the progresses in cryopreservation opened new market opportunities for specimens’ storage and applications: ovules, sperm, embryos, stem cells, seeds and many others. Examples are the increasing number of cell banks and assisted reproductive clinics in the last decades. On the topic of human health, the advances of cryopreservation techniques helps to respond on hospital transplantations demand for tissues and organs. Although all improvements in cryobiology through the last 60 years, nowadays techniques are still not satisfactory for tissue and organs 5. Cryopreservation methods are slow freezing and vitrification, and both aim to overcome ice damage and the presence of solutes in toxic concentrations, which can have a detrimental effect on cell viability during freezing and thawing steps

40,41

. A schematic diagram of the processes

involved in the cryopreservation is presented in figure 3. Slow freezing comprehends the use of cryoprotectants agents (CPA) and slow cooling rates to delocalize water from the cells to the cryopreservation media, were ice will be formed instead of inside the cells

40

. Associated disadvantages are the demanding tight control of the cooling

process to avoid chilling injury, the need of expensive equipment, and it is a time consuming method that generally destroys the extracellular matrix beinguseless for tissues and organs 5,40. In vitrification a high concentration of cryoprotectant agents is added to the medium, thus avoiding the crystallization of water. Because ice formation is avoided there is no need for a slow cooling process, therefore vitrification of the living system is achieved by a fast cooling step below the glass transition temperature 42. Vitrification is defined as a change in the structure of the material between a rubber-like state and a glass-like state. This glass transition is a second order transition that occurs at the glass transition temperature (Tg).

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Fig. 3. Schematic diagram of the processes involved in the cryopreservation either by slow freezing or vitrification.

In 1968, the success of the vitrification process to preserve the biological activity and molecular stability, was proven in human erythrocytes using a concentrated solution of glycerol

43,44

Cryopreservation by vitrification has been growing exponentially since early 80’s and in 1985 Rall and co-workers demonstrated for the first time the vitrification of mouse embryos 45. Today, it is mostly accepted that vitrification performs better than slow freezing as demonstrated in systematic reviews and meta-analysis of human embryos cryopreservation for reproductive medicine

41,46

. Vitrification avoids chilling injury from ice crystals and it does not require

optimal cooling/warming rates due to the thermal behavior which presents no crystallization step 47

. The vitrification process challenges lie mostly on sample size and potential toxic effect of the

high concentration of cryoprotectants. Additionally, the osmotic effects of adding and removing the cryoprotective agents, are still major drawbacks hindering the application of vitrification in the preservation of cells, tissues and organs. Table 2 summarizes the advantages and disadvantages slow freezing versus vitrification.

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Table 2. Summary of the advantages and disadvantages of slow freezing and vitrification processes

Advantages

Slow freezing

Vitrification

Low concentration of CPA (1.5 M)

Shorter exposure time

Low risk of chemical damage

Faster process

Technically simpler

No machine required No ice crystals formed Control of solute penetration

Disadvantages

Longer process

Low concentration of CPA (3-6

Longer exposure time

M)

Freezing machine required

High risk of chemical damage

Ice crystals formed

More clinical expertise

Poor control on solute penetration

Cryoprotectants comprise a large range of molecules, such as sugars, polyalchools, amides, aminoacids, polysaccharides and even macromolecules, such as proteins 4,48. Cryoprotectants can be divided into two categories, the penetrating cryoprotectants which are generally low molecular weight (MW) agents (bellow 100 Da) and the non-penetrating cryoprotectants, namely sugars with a MW in the range 180-600 Da (Figure 4). Other non-penetrating cryoprotectants used are high molecular weight molecules, with MW > 1000 Da, such as ficoll, dextran, polyvinyl pyrrolidone, polyethylene glycol and polyvinyl alcohol 49,50 The cryoprotectant agents’ mechanism of action involves various factors and it is not fully understood. General features of cryoprotectant agents are its viscosity in high concentration and the ability to interact with water molecules by hydrogen bonding

4,5

. Different agents and

mixtures are available but an explanation for the difference in efficiency is not yet recognized. Furthermore, currently used cryoprotectants may have inherent toxicity, limiting the concentrations in which they may be used 42. Glycerol is a polyol that can be found in organisms and it is involved on fatty acids biochemistry 51

. For decades it has been used as a cryoprotectant agent. Since the use of glycerol as a

cryoprotectant agent in vitrification of spermatozoa by Polge and co-workers in the 40’s 52, this has been the standard for the preservation of cell lines, mammalian embryos, and more.

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Although glycerol is considered a low toxic molecule in short-term exposure, it has been reported to damage some tissues 53,54 and the ability to preserve larger human tissues and organs has not yet been discovered.

Fig. 4. Molecular structure of penetrating (MW < 100 Da) and non-penetrating (MW 180-600 Da) cryopreservants.

The introduction of sugars, e.g. sucrose and trehalose, to cryoprotective solutions allow for the concentration reduction of other compounds, namely 2,3-butanediol, to achieve an amorphous state. For example, if 6% w/v of sucrose is added, the concentration of 2,3-butanediol can be reduced about 6% w/v, which can be significant in terms of toxicity 4. Trehalose is a non-reducing disaccharide present in living systems, form animals and plants to yeasts and bacteria. It functions as an energy source, osmoregulator, structural element of bacterial membranes and a stress responsive metabolite during dehydration 55,56. Trehalose, such as other sugar molecules, can be maintained in a vitreous or glass state with a characteristic Tg. Crowe and his group developed diverse studies regarding trehalose role in stabilization of biological samples during dehydration and freezing, however the mechanism is not fully unraveled 57–59. The vitrification of intracellular water by the increased concentration of trehalose or other sugars is a valid hypothesis to explain the mechanism in dry and freezing environments

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8,56,60,61

. In 1991, Koster hypothesized that the presence of a particular combination of sugars in

desiccation-tolerant seeds, which have a glass transition temperature above 0 ºC, is the reason that these seeds are tolerant to desiccation. On the other hand, the sugars present on non-tolerant seeds, present a subzero glass transition temperature

61,62

. Facts contributing for the ability to

achieve a glass state are the tissue dehydration and accumulation of sugar molecules that enhance the system’s viscosity. The presence of trehalose and glycerol mixtures are also known to have effects of the stabilization of enzymes as reported by Barreca et al. 63.

Natural Deep Eutectic Solvents

A notorious wide range of sugar, polyols and aminoacids has been proven to be part of a multicomponent system for the resistance to extreme temperature differences in the animal kingdom. This knowledge was the base for cryoprotectant solutions development in the last century, however this knowledge has not until recently been related with deep eutectic systems. Inspired in nature, Choi and co-workers have related the presence of naturally occurring molecules in plant cells with their cellular metabolism and physiology

64

. These compounds

which are present in large amounts in cells are proposed to form a third type of liquid, one separate from water and lipids, so called natural deep eutectic solvents (NADES) 64. NADES are defined as a mixture of two or more solid or liquid components, such as sugars, aminoacids, organic acids or choline derivatives, which at a particular composition present a high melting point depression, compared to its individual constituents, becoming liquid at room temperature or near room temperature 65. Eutectic solvents have scarcely been reported in the literature until the beginning of the 21st century

66,67

, and the most common are based on choline chloride,

carboxylic acids and other hydrogen-bond donors, e.g. urea, succinic acid and glycerol. Durand et al. has also recognized that deep eutectic solvents are not only present in plants but also in animals

68

. The role of a third liquid phase in organisms’ biochemistry has not yet been fully

explored, however there are some reports in literature that relate the presence of NADES to the ability to transport and dissolve poorly water soluble molecules as well as to improve the activity and stability of enzymes. NADES play an important role in the biotransformation of these poorly

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water soluble molecules, providing a substrate in which both solutes and enzymes can be solubilized 68. Several natural deep eutectic systems have been reported in the literature and the number continues to increase as more information on NADES is unraveled. Certain combinations of the metabolites synthetized by extreme cold-tolerant animals (listed in Table 1) have demonstrated the ability to form NADES. Table 3 presents a summary of the systems reported in the literature where the constituents are metabolites found in extreme cold-tolerant animals. As it can be noticed not many of the systems reported in Table 1 have been described.

Table 3. NADES described in the literature comprising only the metabolites found in extreme cold-tolerant animals

Component 1

Component 2

Component 3

Ratio

Ref

proline

sorbitol

-

1:1

69

proline

glucose

-

5:3

66,69

proline

glucose

-

1:1

68,69

serine

glucose

-

5:4

69

glutamic salt

glucose

-

1:1

69

glucose

frutose

-

1:1

64

urea

sorbitol

NH4Cl

2:7:1

70,71

urea

glucose

CaCl2

4:5:1

71

urea

glucose

CaCl2

3:6:1

72

The peculiar and distinct feature of NADES is the fact that its properties are not the summation of each component characteristics, NADES possesses distinguish features from its pure components. Its chemistry is based in the complexation of a hydrogen bond acceptor and a hydrogen bond donor. The hydrogen bond network formed between the two or more components of the system results in a supermolecular structure 64,73,74. The more distinct characteristic of the NADES system is the reduced melting temperature compared to its individual components. This depression in the melting point is accepted to be consequence of the charge of the hydrogen bond

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network formation

64,65,75

. The molar ratio of the components is hence, critical to have a single

liquid NADES system. Depending of the molar ratio and temperature, the mixture can be above the eutectic point and a single liquid phase composed solely by a supramolecular structure/complex is obtained A two phase system composed of a liquid phase and a solid phase can also be obtained, or in some cases a, single liquid phase can be obtained but composed by the liquid supramolecular structure and by the solid solubilized in it. Naturally, in these cases the properties of the mixture differs from that of a NADES. However, other properties are of great importance, namely, density, polarity and viscosity. NADES density is higher than water and it decreases when water is added to the system

74,76,77

.

In terms of polarity NADES are in general polar systems and their polarity is increased by inclusion of organic acids

73,76

and by the presence of water. In fact, the water percentage can

only be raised until a limit point, after which the hydrogen bonds between the molecules which constitute the NADES are disrupted and the characteristic properties of NADES are lost. For example, experiences with 1,2-propanediol and choline chloride in the molar ratio 1:1 have shown that above 50 wt. % of water the structural supermolecular complex of the NADES system is completely disrupted

73

. The high viscosity is another inherent characteristic of

NADES systems, which is explained by the extensive hydrogen bond interactions between components. The type of hydrogen bond donor, has been associated with the viscosity 74. This property was also found to be modified by the percentage of water in the system, i.e., increasing water content, the viscosity decreases

73,74

the system and cannot be evaporated

64

. Below the critical limit, water in NADES is part of

. Accumulation of NADES can ensure that a minimum

quantity of water molecules is retained in the living being. Overall, NADES are systems with no or low water content and the water percentage extensively alters its properties (density, polarity and viscosity), therefore the water content of the systems is a critical parameter which should be rigorously controlled. The thermal behavior of NADES systems have been study in the last few years. Besides melting, also the thermal decomposition and the glass transition temperature have been studied for NADES. Craveiro and co-workers have shown that NADES are glass formers

76

. The thermal

properties of NADES have been described for different systems and at least one glass transition temperature has been detected. However few studies couple differential scanning calorimetry

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(DSC) with polarized optical microscopy (POM). In the study reported by Craveiro it is possible to observe the glassy state of NADES for the system glucose:tartaric acid in a molar ratio of 1:1. In order to show the influence of NADES in the vitrification process of water, and its impact in the melting and crystallization phenomena, this work shows preliminary experimental data, on studies carried out by adding different amounts of a NADES. In order to perform these studies, a NADES composed of proline and glucose was prepared in a molar ratio of 5:3 (respectively), since this metabolites are often found in animals that live in extreme cold environments (Table 3), and the ability of these compounds to form a NADES has been previously reported 66. Differential scanning calorimetry (DSC) experiments were carried out for the DES composed of proline and glucose with a molar ratio of 5:3, respectively- pro:glc (5:3). This NADES is composed of metabolites found in animals that live in extreme cold environments (Table 2), and its DSC are also presented in Figure 5. The NADES pro:glc (5:3) is liquid at room temperature and the only thermal event presented in the DSC thermogram between -90 and 40 oC is glass transition, with a Tg located at -26 oC (Figure 5), and is very distinct from that of its pure compounds, showing no crystallization or melting events. Figure 6 shows the thermograms of pure water and of water mixed with different amounts of pro:glc (5:3), and shows that the DES has a clear effect on the thermal behavior of water. Pure water (upper left) shows a typical water crystallization peak at ca. -14.9 ºC, which has a distinctive form due to the self-heating of the sample

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and a melting point at -4.3 ºC. As 20 (wt.)% of pro:glc (5:3) is added to water (upper

right) a decrease in the crystallization and melting temperature is observed to -7.1 ºC and -15.1 ºC respectively. When 50 (wt.)% of pro:glc (5:3) is added (lower left) a crystallization event is observed during the cooling cycle, nevertheless it occurs at a significantly lower temperature when compared with crystallization of pure water, since crystalization temperature occurs at 35.9 ºC followed by the respective melting at -20.2 ºC. When higher amounts of pro:glc (5:3) are added to water, more specifically 80 (wt.)% (lower right), no thermal events are observed at the studied temperature range, except for glass transition, thus no crystallization are observed at temperatures above -50 ºC. At these conditions the interactions established between water and the DES are strong enough to maintain water liquid even at such extreme low temperatures.

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Fig. 6. Thermogram obtained at 10 °C·min−1 for pure water and different mixtures of water and the NADES pro:glc in a 5:3 molar ratio – pro:glc (5:3) – 20 wt%, 50 wt% and 80 wt%. DSC thermograms were collected between -90 ºC and 30 ºC. At least 3 heating and cooling cycles are presented.

Additionally it is crucial to study the biocompatibility of NADES towards living beings. The toxicity and biocompatibility of different NADES have been reported in the literature mostly for different choline chloride based systems

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. The results presented demonstrate that NADES

can be biocompatible and non-toxic and therefore could be used as new cryoprotective agents. The understanding of the physico-chemical, and thermal properties of NADES and to couple these knowledge with a thorough study on their biological performance may open new perspectives in vitrification processes of cells, tissues and ultimately organs 81.

Conclusions Cellular water is considered the key of life; however different biological metabolites are synthesized by different organisms to ensure that life is possible below ambient water freezing point, many times using dehydration strategies. The overview of those dehydration strategies under extreme cold conditions has shown an unexpected increased of metabolites, such as sugars, polyols and aminoacids, with increased osmolality and a significant reduction of available free water, concomitant to a body fluid viscosity increment. Moreover, the survival of animals and vitrification of water in the organisms with this cellular biochemistry has been proven, reveling that the systems composed of those metabolites are virtually water-free but can still support life. The possibility of the metabolites to form NADES is an explanation for the

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overwinter natural strategy of survival of some animals. Nature observations were many times used as starting point to successful improvements in a diverse scientific landscape. A better understanding of cryoprotectant agents from extreme cold tolerant animals and an innovative developmental approach based on NADES holds a new path for cryopreservation by vitrification of complex structures, such as tissues and organs, without compromising their function.

Acknowledgments The authors gratefully acknowledge the financial support of FCT through the project Des.zyme Biocatalytic separation of enantiomers using Natural Deep Eutectic Solvents (PTDC/BBBEBB/1676/2014). The funding received from the European Union Seventh Framework Programme (FP7/2007-2013) under grant agreement numbers REGPOT-CT2012-316331POLARIS, as well as from European Regional Development Fund (ERDF) under the project “Novel smart and biomimetic materials for innovative regenerative medicine approaches” RL1ABMR-NORTE-01-0124-FEDER-000016), cofinanced by North Portugal Regional Operational Programme (ON.2, O Novo Norte), under the National Strategic Reference Framework (NSRF) are also appreciatively acknowledged.

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NADES - the key to survive extreme temperature amplitudes - A biomimetic approach towards the development of new cryoprotectants.

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Ana Gertrudes was born in Coimbra in 1986. She is graduated in Biochemistry by University of Coimbra, and completed her MSc in Biochemistry in 2011 by the same university. In 2011, she performed an internship as researcher in drug discovery for the biopharmaceutical Anacor, USA. After it and until 2015, Ana Gertrudes was part of the development team of the Portuguese biotech Stemmatters, for injectable medical devices and combined ATMPs for arthritis. Currently, she performs duties as quality assurance manager in the clinical research organization Blueclinical. Her experience combines knowledge from GMP and GCP working environments, polysaccharides extraction and characterization, primary and immortalized cells manipulation (suspension, 2D and 3D cultures) and characterization. Since her work with combined ATMPs, she became interest on cell preservations techniques that could avoid frozen as solution for stable shelf products with higher viabilities.

Rita Craveiro was born in 1983 in Covilhã. She graduated in chemistry in 2008 from the Universidade de Coimbra and received her MSc in 2010 from the Universidade de Aveiro. She obtained her PhD in Sustainable Chemistry in 2015 from Universidade Nova de Lisboa, in alternative processes for bio-based polymer processing. She is currently a Post-Doc at the Faculdade de Ciências e Tecnologia of the Universidade Nova de Lisboa and associated laboratory LAQV@Requimte, and is involved in various projects and in the co-supervision of two master thesis works. Her main research interests are focused on the area of green chemistry and in the development, characterization and application of deep eutectic solvents in areas that range from polymer processing, biomass processing to biocatalysis and enantiomeric separations. She also works in areas such as supercritical fluids and their applications with eutectic solvents. Currently, she is the author and co-author of several scientific papers, mainly in the area of eutectic solvents, polymer processing and characterization.

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Zahara Eltayari is a 20 year old student from the Faculdade de Ciências e Tecnologia of the Universidade Nova de Lisboa. She is currently studying Chemical and Biochemical Engineering and anything she thinks that might be an opportunity to grow up as a student and as a future professional she likes to participate. She already did three summer internships and attended to several Scientific Conferences. One of the internships she did was in 2016 at the LAQV@Requimte group where she joined the research team to start developing the project about the study of the cryopreservation methods using Natural Deep Eutetic Solvents in the water vitrification process. Zahara is going now to start her master degree and her biggest goal is to specialize in areas such as the Biochemical Engineering, Bioenergetics and Biology. She is also looking forward to do her master thesis somewhere abroad in order to enrich her knowledge and understand a bit more what kind of research projects are being made around the world and what areas she would like to work in the future, as an engineer or as a researcher.

Rui L. Reis, PhD, DSc, Hon. Causa MD, FBSE, FTERM is 50 years. He is the Vice-Rector for R&D of University of Minho (UMinho), Portugal. He is the Director of the 3B’s Research Group and of the ICVS/3B´s PT Government Associate Laboratory of UMinho. He is also the CEO of the European Institute of Excellence on Tissue Engineering and Regenerative Medicine (TERM). He is the World President of TERMIS (Tissue Engineering & Regenerative Medicine International Society) and the editor-in-chief of the Journal of Tissue Engineering and Regenerative Medicine (Wiley-Blackwell). Rui L. Reis education background includes: (i) a graduation in Metal. Eng., U. Porto, Portugal, 1990, (ii) a Master degree by research on Mater. Sci. and Eng. – Biomaterials – obtained in a joint program of the six major technical Universities in Portugal, awarded by U. Porto, Portugal, 1994 (iii) a PhD on Polymer Eng. – Biomaterials & Tissue Engineering, U. Minho, Portugal, degree that was prepared in co-operation with Brunel University, London, UK, 1999, (iv) a Doctor of Science (D.Sc.) degree on Biomedical Engineering - Biomaterials & Tissue Engineering, by U. Minho, Portugal, 2007. He is co-author of 1047 ISI listed publications (855 full papers in scientific journals), with 23850 citations and an hindex of 76, around 250 book chapters, 35 patents and 7 books. He is PI of projects totalizing around 45 MEuros, including the very prestigious European Research Council (ERC) Advanced Grant.

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He was awarded several major national and international scientific and innovation awards, including both the Jean Leray and George Winter Awards from the European Society for Biomaterials (ESB), the Clemson Award (Soc. For Biomaterials, USA) and the TERMIS-EU awards, both for contributions to the Literature and the UNESCO Life Sciences International Award, among many others. He was also awarded a honoris causa degree by the University of Granada, Spain and is a member of the National Academy of Engineering (USA). Rui L. Reis has been involved in biomaterials research since 1990. His main area of research is the development of biomaterials from natural origin polymers (starch, chitin, chitosan, casein, soy, algae based materials, silk fibroin, gellan gum, carragenan, hyaluronic acid, xanthan, marine collagen, etc.) that in many cases his group originally proposed for a range of biomedical applications, including bone replacement and fixation, drug delivery carriers, partially degradable bone cements and tissue engineering scaffolding. Lately the research of his group has been increasingly focused on tissue engineering, regenerative medicine, stem cells and drug delivery applications. His research group works with bone marrow, adipose-derived, umbilical cord (blood and matrix), amniotic origin (fluid and membrane) and embryonic stem cells.

Alexandre Paiva was born in Lisbon, Portugal in 1978. He graduated in Chemical Engineering from the Faculdade de Ciências e Tecnologia of the Universidade Nova de Lisboa in 2001 and completed his PhD in Chemical Engineering – thermodynamics and Biocatalysis from the Technische Universität Hamburg-Harburg in Germany, 2008. From 2010 to 2012 he was an invited professor at the Instituto Piaget were he lectured Introduction to Chemical Engineering and Transport Phenomenon I and II. Since 2008 he is also an assistant lecturer in Biocatalysis. From 2008 to 2016 he was a Post-doc researcher at REQUIMTE, a non-profit scientific organization, working on the “Biodiesel production from green sources using supercritical technology” project. Since 2016 he is a senior researcher at LAQV@REQUIMTE. Alexandre Paiva has more than 30 papers in international scientific peer-reviewed journals. His main areas of expertise are sub-, supercritical fluids, biocatalysis, deep eutectic solvents and the application of these solvents in the valorization of agro-industrial by-products. He is/was involved in 1 Marie Curie project, 4 scientific projects financed by the Portuguese Foundation for Science and Technology, being the principal investigator in 2 of those projects. He is/was also involved in 4 projects in collaboration with Portuguese and international companies. He is the main supervisor of 4 PhDs. and the co-supervisor of 2 more. He is/was the supervisor of more than 20 Master degree thesis.

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Ana Rita C. Duarte was born in Lisbon in 1978. Currently research assistant at the 3B’s research group at Universidade do Minho, She graduated in Chemical Engineering by Faculdade de Ciências e Tecnologia, Universidade Nova de Lisboa in 2002 and completed her PhD on Exploring supercritical fluid technology for the preparation of controlled drug delivery systems in 2006 by the same University. In 2006/2007 she was a researcher at Techniche Universiteit Delft, The Netherlands. From 2007 she was a post-doc at the group where now she currently working, the 3B’s research group. The International Society for Advancement of Supercritical Fluids granted her thesis the Best Thesis Award in 2007. At the moment, she has 79 papers listed in web of knowledge with a total of 1617 citations, and an h-index of 25. Her main research interests are the use of green technologies for the development of biomaterials. In particular, the use of water, ionic liquids, supercritical fluids together with the exploration of natural deep eutectic solvents for natural-based polymer processing are her main scientific interests.

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Graphical abstract

How do animals survive extreme temperature amplitudes? The role of Natural Deep Eutectic Solvents

NADES - the key to survive extreme temperature amplitudes - A biomimetic approach towards the development of new cryoprotectants.

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