This project has received funding from the European Research Council (ERC) under the European Union&#8217;s Horizon 2020 research and innovation program, under grant agreement No ERC-2016-CoG 725034. This work was also supported by the Associate Laboratory for Green Chemistry-LAQV which is financed by national funds from FCT/MCTES (UID/QUI/50006/2019) and by FCT/MCTES through the project CryoDES (PTDC/EQU-EQU/29851/2017).

NOTE: The NADES studied were betaine:L-(+)-tartaric acid (2:1), &#946;-alanine:DL-malic acid (3:2), glucose:sucrose (1:1) and citric acid:glucose (2:1). These systems were prepared by different methods: freeze-drying (FD), vacuum evaporation (VE), and heat and stirring (HS) with and without water. As an example, the protocol for the system citric acid:glucose (2:1) is given. The NADES were characterized by differential scanning calorimetry (DSC), polarized optical microscopy (POM), water content and nuclear magnetic resonance (NMR) spectroscopy.
1. NADES preparation
<ol>
	<li>Freeze drying
	<ol>
		<li>In separate containers, add 2 g of citric acid monohydrate and 0.9530 g of glucose monohydrate. Add 10 mL of deionized water to each and stir until the compounds are completely dissolved.</li>
		<li>Mix the two solutions together and ensure the homogenization of the final solution. Place the solution in a round bottom flask.</li>
		<li>Freeze it using liquid nitrogen. Place the flask in a freeze-dryer for 48 h to ensure that all the water is removed from the sample.</li>
	</ol>
	</li>
	<li>Vacuum evaporation
	<ol>
		<li>Weigh 2 g of citric acid monohydrate and 0.9530 g of glucose monohydrate in separate containers. Add 10 mL of deionized water to each and stir until the compounds are completely dissolved.</li>
		<li>Mix the two solutions together and ensure the homogenization of the solution. Place the solution in a round bottom flask.</li>
		<li>Using a rotary evaporator, dry the sample until a clear, viscous liquid is formed.</li>
	</ol>
	</li>
	<li>Heating and stirring
	<ol>
		<li>Weigh 2 g of citric acid monohydrate and 0.9530 g of glucose monohydrate into the same vial. Add 278 &#956;L of water.</li>
		<li>Place the vial with a magnetic stirring bar in a 50 &#176;C water bath.</li>
		<li>Leave the sample until a clear, viscous liquid is formed.</li>
	</ol>
	</li>
</ol>
2. NADES characterization
<ol>
	<li>Polarized optical microscopy (POM)
	<ol>
		<li>Place a droplet of NADES on a microscope glass slide for observation.</li>
		<li>Using the transmission mode of a microscope, perform the optical characterization of the sample at room temperature.</li>
	</ol>
	</li>
	<li>Karl-Fisher titration
	<ol>
		<li>Collect 100 &#956;L of NADES in a syringe, and then clean the excess liquid on the outside.</li>
		<li>Place the syringe on a scale and tare it.</li>
		<li>Press START on the KF equipment and add a small drop of the sample to the vessel.</li>
		<li>Weigh the syringe, enter the mass on the KF equipment and press ENTER. The result will appear on the screen in ppm of water.</li>
	</ol>
	</li>
	<li>Differential scanning calorimetry (DSC)
	<ol>
		<li>Place 3-10 mg of each sample in a hermetic aluminum pan with a covering lid. Close the pan with a sample press.</li>
		<li>Analyze the samples using a DSC with a temperature range of -90 &#176;C up to the degradation temperature, with a heating rate of 10 &#176;C/min. Perform two cycles with an isothermal hold of 2 min and analyze under a nitrogen atmosphere (50 mL/min).</li>
	</ol>
	</li>
	<li>Nuclear magnetic resonance (NMR)
	<ol>
		<li>Prepare a 5 mm NMR tube by dissolving 250 &#181;L of NADES with 250 &#181;L of dimethyl sulfoxide-d6 (DMSO-d6).</li>
		<li>Acquire the 1H and NOESY spectra at 25 &#176;C on a 400 MHz spectrometer.</li>
		<li>Use an appropriate software to analyze the spectra, and use the chemical shift of DMSO-d6 (&#948; 2.50 ppm) to assign all the signals of each component.</li>
	</ol>
	</li>
</ol>

<ol>
	<li>Paiva, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Natural+deep+eutectic+solvents+-+solvents+for+the+21st+century.">Natural deep eutectic solvents - solvents for the 21st century.</a> ACS Sustainable Chemistry &amp; Engineering. 2, 1063-1071 (2014).</li><li>Abbott, A. P., Capper, G., Davies, D. L., Rasheed, R. K., Tambyrajah, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Novel+solvent+properties+of+choline+chloride/urea+mixtures.">Novel solvent properties of choline chloride/urea mixtures.</a> Chemical Communications. , 70-71 (2003).</li><li>Liu, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Natural+deep+eutectic+solvents:+properties,+applications,+and+perspectives.">Natural deep eutectic solvents: properties, applications, and perspectives.</a> Journal of Natural Products. 81, 679-690 (2018).</li><li>Choi, Y. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Are+natural+deep+eutectic+solvents+the+missing+link+in+understanding+cellular+metabolism+and+physiology.">Are natural deep eutectic solvents the missing link in understanding cellular metabolism and physiology.</a> Plant Physiology. 156, 1701-1705 (2011).</li><li>Dai, Y., Spromsen, J. V., Witkamp, G. -. J., Verpoorte, R., Choi, Y. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Natural+deep+eutectic+solvents+as+new+potential+media+for+green+technology.">Natural deep eutectic solvents as new potential media for green technology.</a> Analytica Chimica Acta. 766, 61-68 (2013).</li><li>Espino, M., Fern&#225;ndez, M. A., Gomez, F. J. V., Silva, M. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Natural+designer+solvents+for+greening+analytical+chemistry.">Natural designer solvents for greening analytical chemistry.</a> Trends in Analytical Chemistry. 76, 126-136 (2016).</li><li>Hayyan, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Natural+deep+eutectic+solvents:+cytotoxic+profile.">Natural deep eutectic solvents: cytotoxic profile.</a> Springer Plus. 5, 913 (2016).</li><li>Dai, Y., Witkamp, G. -. J., Verpoorte, R., Choi, Y. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tailoring+properties+of+natural+deep+eutectic+solvents+with+water+to+facilitate+their+applications.">Tailoring properties of natural deep eutectic solvents with water to facilitate their applications.</a> Food Chemistry. 187, 14-19 (2015).</li><li>Choi, Y. H., Verpoorte, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Green+solvents+for+the+extraction+of+bioactive+compounds+from+natural+products+using+ionic+liquids+and+deep+eutectic+solvents.">Green solvents for the extraction of bioactive compounds from natural products using ionic liquids and deep eutectic solvents.</a> Current Opinion in Food Science. 26, 87-93 (2019).</li><li>Guit&#233;rrez, M. C., Ferrer, M. L., Mateo, C. R., Del Monte, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Freeze-drying+of+aqueous+solutions+of+deep+eutectic+solvents:+a+suitable+approach+to+deep+eutectic+suspensions+of+self-assembled+structures.">Freeze-drying of aqueous solutions of deep eutectic solvents: a suitable approach to deep eutectic suspensions of self-assembled structures.</a> Langmuir. 25, 5509-5515 (2009).</li><li>Gomez, F. J. V., Espino, M., Fern&#225;ndez, M. A., Silva, M. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+greener+approach+to+prepare+natural+deep+eutectic+solvents.">A greener approach to prepare natural deep eutectic solvents.</a> Chemistry Select. 3, 6122-6125 (2018).</li></ol>

The authors have nothing to disclose.

The different methodologies reported in the literature for the preparation of NADES are a heating and stirring method (HS), vacuum evaporation (VE), and freeze-drying (FD). The systems we have prepared in this work are described by different authors in the literature4,5,6,10,11. Table 1 lists the components of each mixture, as reported in the original manuscript as well as their preparation method.
Upon our investigations to reproduce the systems described, we realized that in some cases it was not possible to achieve a similar NADES, as a clear, viscous, liquid sample at room temperature. Preparing a NADES relies on many factors. Some can be easily controlled, but others are more difficult to standardize. The most important thing to consider is that the final product cannot rely on external factors such as the equipment used.
The systems prepared by different methods were then characterized. With polarized optical microscopy (POM), it was observed that with the HS method without water, even at different temperatures, the NADES did not form a clear and viscous liquid. However, a homogeneous and clear, viscous liquid was observed as represented in Figure 1 when applying the HS method with small amounts of water and the VE method for the preparation of the NADES.
DSC was used to determine the thermal events of the mixture. The results showed that the system is liquid at room temperature and up to 130 &#176;C, since the thermogram shows no thermal events. The water content of each sample was measured by Karl-Fischer titration, and the results are represented in Table 2. The water content of the systems must be reported, since it is the parameter that most influences the properties of the obtained liquid, such as viscosity and polarity. These changes have great impact on the outcome of the application for which the NADES is designed.
NMR was also used to confirm the formation the mentioned NADES systems, through the formation of hydrogen bonds between the molecules of each system. One example is given in Figure 2 for the NADES system citric acid:glucose (2:1) with 17% water obtained by HS where the proton spectrum of this NADES and the starting materials (citric acid and glucose) are overlaid (Figure 2a). From this, it is possible to observe changes in the chemical shifts of some protons from each molecule. The major change is the shifting of the OH proton from citric acid. Originally, this signal appears at 5.16 ppm, but this signal shifts to 6.22 ppm because of the formation of hydrogen bonds. This is confirmed by the NOESY spectrum (Figure 2b), where the strong interaction between the OH from citric acid and the remaining protons is visible. A similar interaction was observed for the other NADES systems.
In this study we observed that the description of the preparation method for eutectic systems reported in literature sometimes are incomplete, due to the lack of information regarding the water content of most systems. In the VE method, the water is added by preparing solutions of different components and mixing at a temperature that leads to the formation of eutectic systems; however, we cannot be sure of the minimum required water content. The knowledge of percentage of water needed to form the systems is considered hence, a crucial point that should always be reported, for others to be able to reproduce the preparation of the different eutectic mixtures.
The best method to use is the HS method with water added as it takes less time to prepare, for cases where the water content is already described. However, if this information is not available, the easiest method is the VE method, where all the available water is removed and only the water interacting with the NADES components remains in the system. In any case, researchers should let the systems evaporate for enough time to ensure that free water is removed from the system. This timing is dependent on the equipment and therefore it is not enough to describe in the materials section the duration of the VE method, but the water content has always to be reported.

Deep eutectic solvents have been named the solvents for the 21st century and are considered a new generation of solvents. They are defined as a mixture of two or more chemical compounds at a particular molar ratio to result in a significant decrease in the melting temperature of the individual components, becoming liquid at room temperature1,2,3. In this sense, the preparation of the solvents does not require any chemical reaction and hence the production yield is 100%. In 2011, Choi and co-workers reported the possibility of naturally occurring DES and named them, natural deep eutectic solvents (NADES)3,4,5. NADES can be prepared from different combinations of sugars, amino acids, organic acids and choline derivatives; and these systems prepared from natural components are inherently biocompatible and biodegradable, presenting considerably less toxicity compared to other alternative solvents (e.g., ionic liquids)5,6,7,8. Since 2015, the number of publications in the field has risen exponentially and the possible applications of NADES are very broad3. Even though many manuscripts and reviews have been published, there are fundamental questions that persist, and scientists have not yet found the answer to intriguing questions such as the mechanisms underlying DES formation. Understanding the DES formation mechanism would lead to a consolidated approach towards the development of new systems, rather than the current trial and error approach. Furthermore, the opportunities in the field are growing each day, as consumers become more aware of the sustainability of their products, not only in terms of their end-life but also in terms of processing itself8,9,10. To drive major innovations in the field of deep eutectic solvents, the standardization of the production and characterization methods is first required. The lack of reproducibility of some of the systems reported in the literature was the motivation to develop this work as we faced this issue several times. Herein, we demonstrate the need and crucial importance to accurately describe the materials and methods and show that although the preparation of DES is a simple and straightforward procedure, there are some key aspects (e.g., the presence/amount of water) that must always be discussed.

<table><tbody><tr><td>5 mm NMR tube</td><td>Norell</td><td></td><td></td></tr><tr><td>Acid citric monohydrate</td><td>Sigma-Aldrich</td><td></td><td></td></tr><tr><td>Advance III spectrometer</td><td>Bruker</td><td></td><td></td></tr><tr><td>Deionized water</td><td></td><td></td><td></td></tr><tr><td>dimethyl sulfoxide-d6</td><td>Sigma-Aldrich</td><td></td><td></td></tr><tr><td>DSC Q200</td><td>TA Instruments, USA</td><td></td><td></td></tr><tr><td>Freeze-dryer CHRIST ALPHA 1-4</td><td>Braun Biotec International</td><td></td><td></td></tr><tr><td>Glucose monohydrate</td><td>Cmd chemicals</td><td></td><td></td></tr><tr><td>Karl Fisher Coulometer</td><td>Metrohm</td><td></td><td></td></tr><tr><td>Olympus BX-51 polarized optical microscope</td><td>Olympus</td><td></td><td></td></tr></tbody></table>

preparation of binary and ternary deep eutectic systems

The preparation of deep eutectic systems (DES) is a priori a simple procedure. By definition, two or more components are mixed together at a given molar ratio to form a DES. However, from our experience in the laboratory, there is a need to standardize the procedure to prepare, characterize and report the methodologies followed by different researchers, so that the results published can be reproduced. In this work, we test different approaches reported in the literature to prepare eutectic systems and evaluated the importance of water in the successful preparation of liquid systems at room temperature. These published eutectic systems were composed of citric acid, glucose, sucrose, malic acid, &#946;-alanine, L-tartaric acid and betaine and not all of preparation methods described could be reproduced. However, in some cases, it was possible to reproduce the systems described, with the inclusion of water as a third component of the eutectic mixture.

From the preparation of NADES, the results we expect to obtain are shown on Figure 1. A description of each system is made below. Using the freeze-drying method, the result should be a solid or a very dense paste since all the water is removed from the system. Using the evaporation method, the result should be a clear and viscous liquid. Using the heating and stirring method with the addition of small amounts of water, the result should be a clear and very viscous liquid.
The results obtained from POM can be seen on Figure 1. When a NADES is completely formed, we expect to see a black image, indicating that the sample is completely amorphous and that there are no crystals remaining in the system. The results obtained from KF titration are described in Table 2. Besides the amount of water that is added to the systems, the percentage of water of the final mixture also depends on the water content of the reagents.
Concerning the DSC, the goal of this technique is also to confirm that the system is liquid in the temperature range that it will be applied, so the expected result is to have a thermogram that shows no thermal events on the temperature range of interest (Table 2). The NMR technique is used to confirm the existence of hydrogen bond formation, which is the main characteristic of NADES systems. This can be confirmed by observation of the change in chemical shifts of each signal, and by analysis of the NOESY spectra, that shows spatial and intermolecular correlations (Figure 2).
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Component 1</td>
			<td>Component 2</td>
			<td>Preparation Method</td>
			<td>Reference</td>
		</tr>
		<tr>
			<td>Betaine (Bet)</td>
			<td>L-(+)-Tartaric Acid (LTA)</td>
			<td>Vacuum evaporating (VE)</td>
			<td>Dai et al. (2013)5 and Espino et al. (2016)6</td>
		</tr>
		<tr>
			<td>&#946;-Alanine (&#946;-A)</td>
			<td>DL-Malic Acid (MA)</td>
			<td>Vacuum evaporating (VE)</td>
			<td>Dai et al. (2013)5 and Espino et al. (2016)6</td>
		</tr>
		<tr>
			<td>Glucose (Gluc)</td>
			<td>Sucrose (Suc)</td>
			<td>Freeze-dried (FD)</td>
			<td>Choi et al. (2011)4 and Espino et al. (2016)6</td>
		</tr>
		<tr>
			<td>Citric Acid (CA)</td>
			<td>Glucose (Gluc)</td>
			<td>Freeze-dried (FD)</td>
			<td>Choi et al. (2011)4 and Espino et al. (2016)6</td>
		</tr>
	</tbody>
</table>
Table 1: Systems reported in literature and their preparation method.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td rowspan="2">NADES</td>
			<td rowspan="2">Preparation Method</td>
			<td>Water Content (%)</td>
		</tr>
		<tr>
			<td>Karl Fischer measure</td>
		</tr>
		<tr>
			<td>Bet:LTA (2:1 + 20% water)</td>
			<td>Heating and stirring, adding water</td>
			<td>19.94 &#177; 1.28</td>
		</tr>
		<tr>
			<td>Bet:LTA (2:1)</td>
			<td>Vacuum evaporating</td>
			<td>11.36 &#177; 0.78</td>
		</tr>
		<tr>
			<td>&#946;-A:MA (3:2 + 11% water)</td>
			<td>Heating and stirring, adding water</td>
			<td>11.45 &#177; 0.25</td>
		</tr>
		<tr>
			<td>&#946;-A:MA (3:2)</td>
			<td>Vacuum evaporating</td>
			<td>18.84 &#177; 1.78</td>
		</tr>
		<tr>
			<td>Gluc:Suc (1:1 + 21% water)</td>
			<td>Heating and stirring, adding water</td>
			<td>20.88 &#177; 0.13</td>
		</tr>
		<tr>
			<td>Gluc:Suc (1:1)</td>
			<td>Vacuum evaporating</td>
			<td>22.56 &#177; 0.48</td>
		</tr>
		<tr>
			<td>CA:Gluc (2:1 + 17% water)</td>
			<td>Heating and stirring, adding water</td>
			<td>17.33 &#177; 0.68</td>
		</tr>
		<tr>
			<td>CA:Gluc (2:1)</td>
			<td>Vacuum evaporating</td>
			<td>20.04 &#177; 0.26</td>
		</tr>
	</tbody>
</table>
Table 2: Water content (%) of the systems prepared by different methods.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/60326/60326fig01.jpg" /> 
Figure 1: Representative results of the NADES when prepared by a) freeze-drying, b) vacuum evaporation and c) heating and stirring with addition of water.&#160;The picture shows that when the system is freeze-dried, the result obtained is a crystal since all the water is removed from the mixture whereas when VE and HS methods are used, the amount of water needed for the NADES to form is present and the obtained result is a homogenous liquid at room temperature. <a href="https://www.jove.com/files/ftp_upload/60326/60326fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/60326/60326fig02.jpg" /> 
Figure 2: Polarized optical microscopy of CA:Glu (2:1) prepared by different methods, with cross polarizers (left image) and parallel polarizers (right image) &#8211; 100 &#956;m (10x amplification).&#160;The black images show that the sample is a liquid at room temperature. The FD sample is completely crystalized since the result obtained from this technique was not a liquid. <a href="https://www.jove.com/files/ftp_upload/60326/60326fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/60326/60326fig03.jpg" /> 
Figure 3: a) Overlay of 1H NMR spectra of the (A) NADES system citric acid:glucose:water (2:1:4), (B) glucose, and (C) citric acid; b) NOESY spectrum of the NADES system citric acid:glucose:water (2:1:4).&#160;The overlaid spectra show the difference in chemical shifts of each component upon DES formation, originated by the establishment of hydrogen bonds between them. The NOESY spectrum shows the interaction between the OH proton from citric acid with the remaining protons from both components. <a href="https://www.jove.com/files/ftp_upload/60326/60326fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Preparation of Binary and Ternary Deep Eutectic Systems at JoVE.com

Preparation of Binary and Ternary Deep Eutectic Systems

This protocol aims to standardize the preparation of deep eutectic systems throughout the scientific community so that these systems can be reproduced.

The preparation of deep eutectic systems (DES) is a priori a simple procedure. By definition, two or more components are mixed together at a given molar ...

preparation-of-binary-and-ternary-deep-eutectic-systems

Research

JoVE Journal

Chemistry

11.8K Views.  Universidade Nova de Lisboa. Reproduceerbaarheid van diepe eutectische systemen gevonden in de literatuur is moeilijk wanneer het watergehalte is genegeerd. Deze methode biedt een standaardisatieprotocol voor de ontwikkeling van deze systemen. Deze techniek zal de reproduceerbaarheid van diepe eutectische systeemvoorbereiding mogelijk maken, wat bijdraagt aan coherente resultaten onder de wetenschappelijke gemeenschap.Diepe eutectische systemen hebben bewezen toepassingen op veel verschillende gebieden, waaronder ...

Video: Preparation of Binary and Ternary Deep Eutectic Systems

Controlling the Size, Shape and Stability of Supramolecular Polymers in Water

For aqueous based supramolecular polymers, the simultaneous control over shape, size and stability is very difficult1. At the same time, the ability to do so is highly important in view of a number of applications in functional soft matter including electronics, biomedical engineering, and sensors. In the past, successful strategies to control the size and shape of supramolecular polymers typically focused on the use of templates2,3, end cappers4 or selective solvent techniques5. 
Here we disclose a strategy based on self-assembling discotic amphiphiles that leads to the control over stack length and shape of ordered, chiral columnar aggregates. By balancing electrostatic repulsive interactions on the hydrophilic rim and attractive non-covalent forces within the hydrophobic core of the polymerizing building block, we manage to create small and discrete spherical objects6,7. Increasing the salt concentration to screen the charges induces a sphere-to-rod transition. Intriguingly, this transition is expressed in an increase of cooperativity in the temperature-dependent self-assembly mechanism, and more stable aggregates are obtained. 
For our study we select a benzene-1,3,5-tricarboxamide (BTA) core connected to a hydrophilic metal chelate via a hydrophobic, fluorinated L-phenylalanine based spacer (Scheme 1). The metal chelate selected is a Gd(III)-DTPA complex that contains two overall remaining charges per complex and necessarily two counter ions. The one-dimensional growth of the aggregate is directed by &pi;-&pi; stacking and intermolecular hydrogen bonding. However, the electrostatic, repulsive forces that arise from the charges on the Gd(III)-DTPA complex start limiting the one-dimensional growth of the BTA-based discotic once a certain size is reached. At millimolar concentrations the formed aggregate has a spherical shape and a diameter of around 5 nm as inferred from 1H-NMR spectroscopy, small angle X-ray scattering, and cryogenic transmission electron microscopy (cryo-TEM). The strength of the electrostatic repulsive interactions between molecules can be reduced by increasing the salt concentration of the buffered solutions. This screening of the charges induces a transition from spherical aggregates into elongated rods with a length &gt; 25 nm. Cryo-TEM allows to visualise the changes in shape and size. In addition, CD spectroscopy permits to derive the mechanistic details of the self-assembly processes before and after the addition of salt. Importantly, the cooperativity -a key feature that dictates the physical properties of the produced supramolecular polymers- increases dramatically upon screening the electrostatic interactions. This increase in cooperativity results in a significant increase in the molecular weight of the formed supramolecular polymers in water.

The goal of this experiment is to determine and control the size, shape and stability of self-assembled discotic amphiphiles in water. For aqueous based supramolecular polymers such level of control is very difficult. We apply a strategy using both repulsive and attractive interactions. The experimental techniques applied to characterize this system are broadly applicable.

For aqueous based supramolecular polymers, the simultaneous control over shape, size and stability is very difficult1. At the same time, the ability to do ...

Engineering

A Facile and Efficient Approach for the Production of Reversible Disulfide Cross-linked Micelles

Nanomedicine is an emerging form of therapy that harnesses the unique properties of particles that are nanometers in scale for biomedical application. Improving drug delivery to maximize therapeutic outcomes and to reduce drug-associated side effects are some of the cornerstones of present-day nanomedicine. Nanoparticles in particular have found a wide application in cancer treatment. Nanoparticles that offer a high degree of flexibility in design, application, and production based on the tumor microenvironment are projected to be more effective with rapid translation into clinical practice. The polymeric micellar nano-carrier is a popular choice for drug delivery applications.
In this article, we describe a simple and effective protocol for synthesizing drug-loaded, disulfide cross-linked micelles based on the self-assembly of a well-defined amphiphilic linear-dendritic copolymer (telodendrimer, TD). TD is composed of polyethylene glycol (PEG) as the hydrophilic segment and a thiolated cholic acid cluster as the core-forming hydrophobic moiety attached stepwise to an amine-terminated PEG using solution-based peptide chemistry. Chemotherapy drugs, such as paclitaxel (PTX), can be loaded using a standard solvent evaporation method. The O2-mediated oxidation was previously utilized to form intra-micellar disulfide cross-links from free thiol groups on the TDs. However, the reaction was slow and not feasible for large-scale production. Recently, an H2O2-mediated oxidation method was explored as a more feasible and efficient approach, and it was 96 times faster than the previously reported method. Using this approach, 50 g of PTX-loaded, disulfide cross-linked nanoparticles have been successfully produced with narrow particle size distribution and high drug loading efficiency. The stability of the resulting micelle solution is analyzed using disrupting conditions such as co-incubation with a detergent, sodium dodecyl sulfate, with or without a reducing agent. The drug-loaded, disulfide cross-linked micelles demonstrated less hemolytic activity when compared to their non-cross-linked counterparts.

To deliver cancer drugs to tumor sites with high specificity and reduced side effects, new methods based on nanoparticles are required. Here, we describe disulfide cross-linked micelles that can be easily prepared by hydrogen peroxide-mediated oxidation and are able to dissociate efficiently under a reducing tumor environment to release payloads.

Nanomedicine is an emerging form of therapy that harnesses the unique properties of particles that are nanometers in scale for biomedical application. ...

Biochemistry

Highly Stereoselective Synthesis of 1,6-Ketoesters Mediated by Ionic Liquids: A Three-component Reaction Enabling Rapid Access to a New Class of Low Molecular Weight Gelators

In organic chemistry ionic liquids (ILs) have emerged as safe and recyclable reaction solvents. In the presence of a base ILs can be deprotonated to form catalytically active N-Heterocyclic Carbenes (NHCs). Here we have used ILs as precatalysts in the addition of &#945;,&#946;-unsaturated aldehydes to chalcones to form 1,6-ketoesters, incorporating an anti-diphenyl moiety in a highly stereoselective fashion. The reaction has a broad substrate scope and several functional groups and heteroaromatics can be integrated into the ketoester backbone in generally good yields with maintained stereoselectivity. The reaction protocol is robust and scalable. The starting materials are inexpensive and the products can be obtained after simple filtration, avoiding solvent-demanding chromatography. Furthermore, the IL can be recycled up to 5 times without any loss of reactivity. Moreover, the 1,6-ketoester end product is a potent gelator in several hydrocarbon based solvents. The method enables rapid access to and evaluation of a new class of low molecular weight gelators (LMWGs) from recyclable and inexpensive starting materials.

Ionic liquids (ILs) mediate fast, simple and cheap access to 1,6-ketoesters in high diastereoselectivities and good yields. The reaction protocol is robust and the 1,6-ketoesters can be obtained in gram scale after a simple filtration protocol. Moreover, the 1,6-ketoesters are potent gelators in hydrocarbon solvents.

In organic chemistry ionic liquids (ILs) have emerged as safe and recyclable reaction solvents. In the presence of a base ILs can be deprotonated to form ...

Construction and Systematical Symmetric Studies of a Series of Supramolecular Clusters with Binary or Ternary Ammonium Triphenylacetates

Functions of clusters in nano or sub-nano scale significantly depend on not only kinds of their components but also arrangements, or symmetry, of their components. Therefore, the arrangements in the clusters have been precisely characterized, especially for metal complexes. Contrary to this, characterizations of molecular arrangements in supramolecular clusters composed of organic molecules are limited to a few cases. This is because construction of the supramolecular clusters, especially obtaining a series of the supramolecular clusters, is difficult due to low stability of non-covalent bonds compare to covalent bonds. From this viewpoint, utilization of organic salts is one of the most useful strategies. A series of the supramolecules could be constructed by combinations of a specific organic molecule with various counter ions. Especially, primary ammonium carboxylates are suitable as typical examples of supramolecules because various kinds of carboxylic acids and primary amines are commercially available, and it is easy to change their combinations. Previously, it was demonstrated that primary ammonium triphenylacetates using various kinds of primary amines specifically construct supramolecular clusters, which are composed of four ammoniums and four triphenylacetates assembled by charge-assisted hydrogen bonds, in crystals obtained from non-polar solvents. This study demonstrates an application of the specific construction of the supramolecular clusters as a strategy to conduct systematical symmetric study for clarification of correlations between molecular arrangements in supramolecules and kinds and numbers of their components. In the same way with binary salts composed of triphenylacetates and one kind of primary ammoniums, ternary organic salts composed of triphenylacetates and two kinds of ammoniums construct the supramolecular clusters, affording a series of the supramolecular clusters with various kinds and numbers of the components.

This article describes construction of a series of hydrogen-bonding supramolecular clusters in crystals using primary ammonium triphenylacetates, which are recrystallized from non-polar solvents. This selective construction of the supramolecular clusters leads to effective systematical symmetric studies about a correlation between the supramolecular clusters and their components.

Functions of clusters in nano or sub-nano scale significantly depend on not only kinds of their components but also arrangements, or symmetry, of their ...

Facile Preparation of Internally Self-assembled Lipid Particles Stabilized by Carbon Nanotubes

We present a facile method to prepare nanostructured lipid particles stabilized by carbon nanotubes (CNTs). Single-walled (pristine) and multi-walled (functionalized) CNTs are used as stabilizers to produce Pickering type oil-in-water (O/W) emulsions. Lipids namely, Dimodan U and Phytantriol are used as emulsifiers, which in excess water self-assemble into the bicontinuous cubic Pn3m phase. This highly viscous phase is fragmented into smaller particles using a probe ultrasonicator in presence of conventional surfactant stabilizers or CNTs as done here. Initially, the CNTs (powder form) are dispersed in water followed by further ultrasonication with the molten lipid to form the final emulsion. During this process the CNTs get coated with lipid molecules, which in turn are presumed to surround the lipid droplets to form a particulate emulsion that is stable for months. The average size of CNT-stabilized nanostructured lipid particles is in the submicron range, which compares well with the particles stabilized using conventional surfactants. Small angle X-ray scattering data confirms the retention of the original Pn3m cubic phase in the CNT-stabilized lipid dispersions as compared to the pure lipid phase (bulk state). Blue shift and lowering of the intensities in characteristic G and G&#39; bands of CNTs observed in Raman spectroscopy characterize the interaction between CNT surface and lipid molecules. These results suggest that the interactions between the CNTs and lipids are responsible for their mutual stabilization in aqueous solutions. As the concentrations of CNTs employed for stabilization are very low and lipid molecules are able to functionalize the CNTs, the toxicity of CNTs is expected to be insignificant while their biocompatibility is greatly enhanced. Hence the present approach finds a great potential in various biomedical applications, for instance, for developing hybrid nanocarrier systems for the delivery of multiple functional molecules as in combination therapy or polytherapy.

We report on a smart application of carbon nanotubes for kinetic stabilization of lipid particles that contain self-assembled nanostructures in their cores. The preparation of lipid particles requires rather low concentrations of carbon nanotubes permitting their use in biomedical applications such as drug delivery.

We present a facile method to prepare nanostructured lipid particles stabilized by carbon nanotubes (CNTs). Single-walled (pristine) and multi-walled ...

Determination of Thermodynamic Properties of Alkaline Earth-liquid Metal Alloys Using the Electromotive Force Technique

A novel electrochemical cell based on a CaF2 solid-state electrolyte has been developed to measure the electromotive force (emf) of binary alkaline earth-liquid metal alloys as functions of both composition and temperature in order to acquire thermodynamic data. The cell consists of a chemically stable solid-state CaF2-AF2 electrolyte (where A is the alkaline-earth element such as Ca, Sr, or Ba), with binary A-B alloy (where B is the liquid metal such as Bi or Sb) working electrodes, and a pure A metal reference electrode. Emf data are collected over a temperature range of 723 K to 1,123 K in 25 K increments for multiple alloy compositions per experiment and the results are analyzed to yield activity values, phase transition temperatures, and partial molar entropies/enthalpies for each composition.

This protocol describes the measurement of the electromotive force of alkaline-earth elements in liquid metal alloys at high temperatures (723-1,123 K) to determine their thermodynamic properties, including activity, partial molar entropy, partial molar enthalpy, and phase transition temperatures, over a wide composition range.

A novel electrochemical cell based on a CaF2 solid-state electrolyte has been developed to measure the electromotive force (emf) of binary alkaline ...

Preparation of Monodomain Liquid Crystal Elastomers and Liquid Crystal Elastomer Nanocomposites

LCEs are shape-responsive materials with fully reversible shape change and potential applications in medicine, tissue engineering, artificial muscles, and as soft robots. Here, we demonstrate the preparation of shape-responsive liquid crystal elastomers (LCEs) and LCE nanocomposites along with characterization of their shape-responsiveness, mechanical properties, and microstructure. Two types of LCEs &#8212; polysiloxane-based and epoxy-based &#8212; are synthesized, aligned, and characterized. Polysiloxane-based LCEs are prepared through two crosslinking steps, the second under an applied load, resulting in monodomain LCEs. Polysiloxane LCE nanocomposites are prepared through the addition of conductive carbon black nanoparticles, both throughout the bulk of the LCE and to the LCE surface. Epoxy-based LCEs are prepared through a reversible esterification reaction. Epoxy-based LCEs are aligned through the application of a uniaxial load at elevated (160 &#176;C) temperatures. Aligned LCEs and LCE nanocomposites are characterized with respect to reversible strain, mechanical stiffness, and liquid crystal ordering using a combination of imaging, two-dimensional X-ray diffraction measurements, differential scanning calorimetry, and dynamic mechanical analysis. LCEs and LCE nanocomposites can be stimulated with heat and/or electrical potential to controllably generate strains in cell culture media, and we demonstrate the application of LCEs as shape-responsive substrates for cell culture using a custom-made apparatus.

We demonstrate the preparation of siloxane-based and epoxy-based liquid crystal elastomers (LCEs) and LCE nanocomposites. The LCEs are characterized with respect to reversible strain, liquid crystal ordering, and stiffness. As a potential application, we demonstrate their use as shape-responsive substrates in a custom device for active cell culture.

LCEs are shape-responsive materials with fully reversible shape change and potential applications in medicine, tissue engineering, artificial muscles, and ...

Bioengineering

Synthesis of Biocompatible Liquid Crystal Elastomer Foams as Cell Scaffolds for 3D Spatial Cell Cultures

Here, we present a step-by-step preparation of a 3D, biodegradable, foam-like cell scaffold. These scaffolds were prepared by cross-linking star block co-polymers featuring cholesterol units as side-chain pendant groups, resulting in smectic-A (SmA) liquid crystal elastomers (LCEs). Foam-like scaffolds, prepared using metal templates, feature interconnected microchannels, making them suitable as 3D cell culture scaffolds. The combined properties of the regular structure of the metal foam and of the elastomer result in a 3D cell scaffold that promotes not only higher cell proliferation compared to conventional porous templated films, but also better management of mass transport (i.e., nutrients, gases, waste, etc.). The nature of the metal template allows for the easy manipulation of foam shapes (i.e., rolls or films) and for the preparation of scaffolds of different pore sizes for different cell studies while preserving the interconnected porous nature of the template. The etching process does not affect the chemistry of the elastomers, preserving their biocompatible and biodegradable nature. We show that these smectic LCEs, when grown for extensive time periods, enable the study of clinically relevant and complex tissue constructs while promoting the growth and proliferation of cells.

This study presents a methodology to prepare 3D, biodegradable, foam-like cell scaffolds based on biocompatible side-chain liquid crystal elastomers (LCEs). Confocal microscopy experiments show that foam-like LCEs allow for cell attachment, proliferation, and the spontaneous alignment of C2C12s myoblasts.

Here, we present a step-by-step preparation of a 3D, biodegradable, foam-like cell scaffold. These scaffolds were prepared by cross-linking star block ...

Rapid, Scalable Assembly and Loading of Bioactive Proteins and Immunostimulants into Diverse Synthetic Nanocarriers Via Flash Nanoprecipitation

Nanomaterials present a wide range of options to customize the controlled delivery of single and combined molecular payloads for therapeutic and imaging applications. This increased specificity can have significant clinical implications, including decreased side effects and lower dosages with higher potency. Furthermore, the in situ targeting and controlled modulation of specific cell subsets can enhance in vitro and in vivo investigations of basic biological phenomena and probe cell function. Unfortunately, the required expertise in nanoscale science, chemistry and engineering often prohibit laboratories without experience in these fields from fabricating and customizing nanomaterials as tools for their investigations or vehicles for their therapeutic strategies. Here, we provide protocols for the synthesis and scalable assembly of a versatile non-toxic block copolymer system amenable to the facile formation and loading of nanoscale vehicles for biomedical applications. Flash nanoprecipitation is presented as a methodology for rapid fabrication of diverse nanocarriers from poly(ethylene glycol)-bl-poly(propylene sulfide) copolymers. These protocols will allow laboratories with a wide range of expertise and resources to easily and reproducibly fabricate advanced nanocarrier delivery systems for their applications. The design and construction of an automated instrument that employs a high-speed syringe pump to facilitate the flash nanoprecipitation process and to allow enhanced control over the homogeneity, size, morphology and loading of polymersome nanocarriers is described.

Nanomaterials provide versatile mechanisms of controlled therapeutic delivery for both basic science and translational applications, but their fabrication often requires expertise that is unavailable in most biomedical laboratories. Here, we present protocols for the scalable fabrication and therapeutic loading of diverse self-assembled nanocarriers using flash nanoprecipitation.

Nanomaterials present a wide range of options to customize the controlled delivery of single and combined molecular payloads for therapeutic and imaging ...

Versatile Biomolecular Condensates from Amphiphilic DNA Nanostars or C-Stars

Synthetic Condensates and Cell-Like Architectures from Amphiphilic DNA Nanostructures

Synthetic droplets and condensates are becoming increasingly common constituents of advanced biomimetic systems and synthetic cells, where they can be used to establish compartmentalization and sustain life-like responses. Synthetic DNA nanostructures have demonstrated significant potential as condensate-forming building blocks owing to their programmable shape, chemical functionalization, and self-assembly behavior. We have recently demonstrated that amphiphilic DNA "nanostars", obtained by labeling DNA junctions with hydrophobic moieties, constitute a particularly robust and versatile solution. The resulting amphiphilic DNA condensates can be programmed to display complex, multi-compartment internal architectures, structurally respond to various external stimuli, synthesize macromolecules, capture and release payloads, undergo morphological transformations, and interact with live cells. Here, we demonstrate protocols for preparing amphiphilic DNA condensates starting from constituent DNA oligonucleotides. We will address (i) single-component systems forming uniform condensates, (ii) two-component systems forming core-shell condensates, and (iii) systems in which the condensates are modified to support in vitro transcription of RNA nanostructures.

Author Spotlight: Developing Synthetic Cells from Programmable Amphiphilic DNA Nanostructures

We present a protocol for preparing synthetic biomolecular condensates consisting of amphiphilic DNA nanostars starting from their constituent DNA oligonucleotides. Condensates are produced from either a single nanostar component or two components and are modified to sustain in vitro transcription of RNA from an embedded DNA template.

Synthetic droplets and condensates are becoming increasingly common constituents of advanced biomimetic systems and synthetic cells, where they can be ...

Preparation of Binary and Ternary Deep Eutectic Systems

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