Comparison of solvate ionic liquids and DMSO as an in vivo delivery and storage media for small molecular therapeutics

Comparison of solvate ionic liquids and DMSO as an in vivo delivery and storage media for small... Background: Solvate ionic liquids (SILs) are a new class of ionic liquids that are equimolar solutions of lithium bistrifluoromethanesulfonimide in either triglyme or tetraglyme, referred to as G3LiTFSA and G4LiTFSA, respectively. SILs play a role in energy storage lithium batteries, and have been proposed as potential alternatives to traditional organic solvents such as DMSO. G3TFSA and G4TFSA have been shown to exhibit no toxicity in vivo up to 0.5% (v/ v), and solubilize small compounds (N,N-diethylaminobenzaldehyde) with full penetrance, similar to DMSO delivered DEAB. Herein, we compare the effects of storage (either at room temperature or − 20 °C) on DEAB solubilized in either DMSO, G3TFSA or G4TFSA to investigate compound degradation and efficacy. Results: The findings show that DEAB stored at room temperature (RT) for 4 months solubilized in either G3TFSA, G4TFSA or DMSO displayed no loss of penetrance. The same was observed with stock solutions stored at − 20 °C for 4 months; however G4TFSA remained in a liquid state compared to both G3TFSA and DMSO. Moreover, we examined the ability of G3TFSA and G4TFSA to solubilize another small molecular therapeutic, the FGFR antagonist SU5402. G4TFSA, unlike G3TFSA solubilized SU5402 and displayed similar phenotypic characteristics and reduced dlx2a expression as reported and shown with SU5402 in DMSO; albeit more penetrative. Conclusion: This study validates the use of these ionic liquids as a potential replacement for DMSO in vivo as organic solubilizing agents. Keywords: Zebrafish, Embryogenesis, Retinoic acid, Aldh1a2, FGFR, DEAB, SU5402, Ionic liquids Background pharmaceutical ingredients to ionic liquid-like salts, typic- In recent years, there has been an increase in the interest ally by inclusion of a charge diffuse cation or anion, which of ionic liquids (ILs) due to their potential in a myriad of can result in improved therapeutic effect via changes in chemical processes. Their unique property of being mol- crystal structure [18, 19]. These approaches have largely ten salts at room temperature imparts unusual properties revolved around the use of imidazolium-derived ionic liq- such as; high ionic conductivity, non-flammability, and uids which are a well-used and studied class of solvents. negligible vapour pressure. Due to these properties, and Of particular interest has been a new class of ionic their high customisability through anion/cation pairing, liquids, termed ‘solvate ionic liquids’ (SILs), reported by have become a staple material used throughout a variety Watanabe et al. [20–26]. The preparation of SILs is of disciplines [1–9]. The use of imidazolium ionic liquids trivial, being simple dissolution of LiNTf (lithium in drug delivery has seen some success, as they offer the bistrifluoromethanesulfonimide) in either triglyme potential to deliver sparingly soluble molecules via oral, or (triethylene glycol dimethyl ether, G3) or tetraglyme (tet- transdermal routes over a long period of time [10–17]. raethylene glycol dimethyl ether, G4) yields the ionic liq- Complementing this effect is the conversion of active uids, G3TFSA or G4TFSA, respectively (Fig. 1). Recent work by our group, and others, has characterised these * Correspondence: y.gibert@deakin.edu.au solvate ionic liquids using Kamlet-Taft parameters, and Metabolic Genetic Diseases Laboratory, Metabolic Research Unit, Deakin explored their use as a solvent for organic chemical University School of Medicine, 75 Pigdons Road, Geelong, VIC 3216, Australia transformations [27, 28]. Recently, we demonstrated the Full list of author information is available at the end of the article © The Author(s). 2018 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Yoganantharajah et al. BMC Biotechnology (2018) 18:32 Page 2 of 7 Fig. 1 Solvate ionic liquids and DMSO which are the focus of this study toxicity of solvate ionic liquids in vivo using a zebrafish G4TFSA, and their ability to solubilize and deliver other (Danio rerio), and found that both G3TFSA and small molecules/pharmacological compounds. G4TFSA with concentrations up to 50 μM (or 0.5%) are not toxic to zebrafish embryos (which are more sensitive to toxicity than adults) [29]. Since most organic modifier Results solvents (such as DMSO) are used at a much lower con- Evaluation of SILs stored at -20 °C centration (usually 0.1%), our study was able to conclude To be consistent with our previous study, which used the that both G3TFSA and G4TFSA can be safely used as retinoic acid synthesis inhibitor 4-diethylaminobenzaldehyde aqueous modifier solvents to allow evaluation of small (DEAB), we continued to use this molecule as the initial molecules. Both G3TFSA and G4TFSA do not induce focus of this work. DEAB is a known inhibitor of retinalde- apoptosis at a similar concentration to DMSO (10 μM) hyde dehydrogenases ALDH1A1, ALDH1A2, and and display a full drug penetrance and the anticipated ALDH1A3 in mammals (teleost fish do not have an aldh1a1 physiological changes induced in the test specimens gene) [34]. Retinaldehyde dehydrogenases convert retinalde- [29]. Since these novel SILs were able to replace DMSO hyde, a product from retinol (Vitamin A1), into retinoic acid as organic modifiers, we were curious if they could be (RA) [35]. Hence, inhibiting the function of retinaldehyde used as an alternative long term storage media for these dehydrogenases via DEAB abolishes the synthesis of RA molecules, and if compound degradation over this required for normal growth and development [36]. Thus, period was reduced compared to DMSO. we wanted to evaluate G3TFSA and G4TFSA’s ability to Currently, the most commonly used organic solvent, keep DEAB stable at RT without compound degradation or in academic and industrial research laboratories, to loss of efficacy. The rationale behind this test is that stock solubilize small organic molecules in water is DMSO solutions kept at RT would remain in a liquid state with no [30]. Due to its ability to dissolve many kinds of com- concern for sample degradation from multiple freeze/thaw pounds, DMSO plays a pivotal role in sample manage- cycles. This was evaluated over a duration of 4 months, and ment and high-throughput screening during in vivo and differences in penetrance of the drug, measured by the in vitro evaluation. strength of the phenotypes were determined. Due to its broad solubilizing capability and apparent To conduct this comparison, N,N-diethylaminobenzal- low toxicity at concentrations < 10%, [31, 32] DMSO is dehyde (DEAB), was administered in parallel to zebrafish used as a solvent for many drug types and is used as the embryos: one sample containing DMSO only (0.1%), one vehicle control of choice for both in vitro and in vivo DEAB in DMSO and the other two, containing DEAB in studies. However, in a study coordinated by Corderio either G3TFSA or G4TFSA. Zebrafish embryos were et al., the authors demonstrated low-dose toxicity of exposed to DEAB in the respective solvents at the begin- DMSO and concluded that solvents other than DMSO ning of gastrulation occurring at 6 h post fertilization should be employed for solubilizing drugs [33]. Thus (hpf) at a final concentration of 5 μM. there is a need to find a suitable replacement for DMSO Comparison of the treatments kept at RT showed that which do not possess this inherent toxicity. zebrafish embryos exposed to DEAB solubilised in either Therefore, the focus and aim of this study was to DMSO (Fig. 2b), G3TFSA (Fig. 2c), or G4TFSA (Fig. 2d) evaluate the performance of the ILs G3TFSA and from 6 to 30 hpf displayed characteristics associated G4TFSA as a potential replacement to the conventional with the loss of RA signalling compared to the control solvent DMSO, using zebrafish as a model organism. (Fig. 2a): [29, 37, 38] lack of pectoral fin induction Since, we have shown the ability of G3TFSA and (arrowhead), shortening of the posterior head (marked G4TFSA to solubilize DEAB with full penetrance of by an asterisk) malformation of the otic vesicle (arrow) reported phenotypic characteristics [29]; we wanted to and pericardiac oedema. There was no discernible differ- evaluate the impact of storage on both G3TFSA and ences in DEAB penetrance between DEAB in either Yoganantharajah et al. BMC Biotechnology (2018) 18:32 Page 3 of 7 embryos treated with 5 μM showed that zebrafish em- bryos exposed to DEAB solubilised in either DMSO (Fig. 3b), G3TFSA (Fig. 3c), or G4TFSA (Fig. 3d) from 6 to 30 hpf displayed the characteristics associated with the loss of RA signalling compared to the control (Fig. 3a)[29, 37, 38] (100% penetrance for all com- pounds tested, n =60). As previously observed in the RT test, there was an observed phenotype that clearly showed a lack of pec- toral fin induction (arrowhead), shortening of the poster- ior head (marked by an asterisk), malformation of the otic vesicle (arrow) and pericardiac oedema. However, unlike both DMSO and G3TFSA, G4TFSA did not freeze at − 20 °C but remained in a viscous state. Fig. 2 Room temperature DEAB exposure in developing zebrafish Capacity of the SILs to deliver small compounds embryos. Embryos exposed to DEAB at 5 μM in solution at room With these data in hand, our attention turned to demon- temperature for 4 months in either DMSO (b), G3TFSA (c), or G4TFSA (d) displayed the reported characteristic of loss of RA (compared to strating the generality of these ionic liquids as a storage control (DMSO exposed only (a): lack of pectoral fin induction and delivery media for small molecular therapeutics. For (arrowhead), shortening of the posterior head (asterisk), the purpose of this study, we evaluated the ability of malformation of the otic vesical (arrow) and pericardial edema both G3TFSA and G4TFSA to solubilise and compare the performance of SU5402 (Fig. 4d) a pan-fibroblast DMSO, G3TFSA or G4TFSA (100% for each, n = 60) growth factor receptors (FGFR) specific tyrosine kinase after storage at RT for 4 months [29]. inhibitor and is used in a multitude of zebrafish develop- mental systems to specifically inhibit FGFR signalling Evaluation of SILs stored at room temperature against DMSO [39–43]. A comparison of zebrafish em- To assess the solvate properties of G3TFSA and bryos treated with both SU5402 in G4TFSA at concen- G4TFSA, it was also imperative to evaluate the perform- trations 2.5 μM (Fig. 4c) and 5 μM (Fig. 4f) from 6 to 30 ance of the ionic liquids G3TFSA and G4TFSA after hpf displayed previously reported phenotypes, such as storage in the conventional (frozen) manner. Therefore, lack of pectoral fin induction (arrowhead) malformation DEAB was stored at − 20 °C for 4 months in either of the otic vesicle (arrow) compared to the control em- DMSO, G3TFSA or G4TFSA. A comparison of zebrafish bryo (Fig. 4a). Embryos treated with SU5402 in DMSO (Fig. 4b) also reported these phenotypes. There was also an evident lack of a formed mid-hindbrain boundary at concentrations of only 5 μM DEAB in DMSO and G4TFSA (open arrowhead) (Fig. 4e, f)[38, 44]. Assessing penetrance of the SILs In addition, we wanted to the examine genes that are affected by FGF (fibroblast growth factor) signalling. Hence, we investigated the effect on dlx2a expression using whole mount in situ hybridisation (WISH) after exposure to 2.5 μM SU5402 that had been either solubi- lized in DMSO or G4TFSA (Fig. 5). WISH results showed that compared to the control embryos (Fig. 5a, d), embryos treated with SU5402 solubilized in both DMSO (Fig. 5b, e) and G4TFSA (Fig. 5c, f) depicted an absence of dlx2a expression in the ventral cranial neural Fig. 3 Frozen DEAB exposure in developing zebrafish embryos. crest cells (arrows) marked by an asterix. However, Embryos exposed to DEAB at 5 μM that have been stored for comparatively there was a greater reduction in the 4 months at -20 °C in either DMSO (b), G3TFSA (c), or G4TFSA (d) display the reported characteristics of loss of RA (compared to expression of dlx2a (marked by AP staining) in the tel- control (DMSO exposed only (a): lack of pectoral fin induction encephalon (arrowhead) in zebrafish embryos that were (arrowhead), shortening of the posterior head (asterisk), treated with SU5402 solubilized in G4TFSA (Fig. 5c, f) malformation of the otic vesical (arrow) and pericardial edema compared to those treated with SU5402 solubilized in Yoganantharajah et al. BMC Biotechnology (2018) 18:32 Page 4 of 7 Fig. 4 SU5402 exposure in developing zebrafish embryos. Embryos are exposed to SU5402 (d)at2.5 μM(b & c)or5 μM(e & f) in either DMSO or G4TFSA display the reported characteristics of FGF signaling inhibition compared to control (untreated (a) or DMSO exposed only (b & e)): lack of pectoral fin induction (marked by an asterisk), malformation of the otic vesicle (arrow). Lloss of MHB (open arrow head) was observed in embryos treated with 5 μM DEAB. 100× magnification DMSO (Fig. 5b, e). This indicates that G4TFSA has as solutions in an environment of low relative humidity, to greater ability to deliver SU5402 (penetrative power) mitigate or retard compound degradation [45]. A study compared to DMSO. conducted by Kozikowski et al., investigating the effect of room temperature storage on the stability of com- Discussion pounds in DMSO concluded that the relationship be- Previous studies have shown that the prolonged storage tween length of storage and the probability of observing of organic compounds in solution can lead to significant the compound is described by a repeated measures sample degradation, and subsequently an increase in the logistic regression model [45]. Results from the study number of false positives for high-throughput biological determined that the probability of observing the com- screening assays [45]. These false positives represent po- pound was 92% after 3 months of storage at room tentially erroneous investment of time and money elab- temperature, 83% after 6 months, and 52% after 1 year orating on a false lead compound. As a result, many in DMSO [45]. Hence, it is valuable to assess the long researchers and pharmaceutical organizations now store term effects of RT storage on DEAB penetrance post their organic compounds as frozen DMSO stock solubilisation in both G3TFSA and GF4TFSA. Fig. 5 Expression of dlx2a visualized using whole mount in situ hybridization. Embryos exposed to SU5402 at 2.5 μM in either DMSO (b & e)or G4TFSA (c & f) display reduced localization and expression of dlx2a compared to control (DMSO exposed only (a & d) in the hindbrain (arrow) and in the pharyngeal arches (arrow head, * lack of pharyngeal arches). The first row depicts the embryos in a later orientation, the second row depicts the embryos in a ventral orientation Yoganantharajah et al. BMC Biotechnology (2018) 18:32 Page 5 of 7 Furthermore, a key characteristic of DMSO is that A previous study by Gibert et al., reported the forma- compared to G4TFSA it has a relatively higher melting tion of oral and pharyngeal dentition in teleosts depends point (T ) of around 19 °C [46], hence DMSO freezes on differential recruitment of retinoic acid signalling and easily and remelts slowly at room temperature. This that a lack of FGF signalling affects the expression of means that if stored frozen, a considerable amount of dlx2a (distal-less homeobox 2a) [49]. Dlx genes are time will be spent getting DMSO (and the compound expressed in a coordinate manner which create proximal solubilized in DMSO) to a liquid state before it can be to distal polarity within the pharyngeal arches [50]. In zeb- used. Whereas, G4TFSA was able to remain in a liquid rafish, dlx2a is expressed in the migrating cranial neural like state at − 20 °C, proving advantageous over DMSO which contributes to the pharyngeal arches [49–53]. as it can be used straight away after being removed from In order to get a more accurate understanding into storage at − 20 °C. Similar to DMSO, G3TFSA albeit whether these SILs can be used clinically we will need to higher, has a similar T of 23 °C [47]. However, G3TFSA continue our studies in adult and mammalian models. has a much higher entropy change of fusion value of Even though zebrafish provide an adequate starting − 1 − 1 112.5 J K mol compared to DMSO which only has an point, the efficacy and penetrance of G3TFSA and − 1 − 1 entropy change of fusion value of around 50 J K mol G4TFSA would need to be assessed in a mammalian [47]. This means that even though both G3TFSA and model which would share more homology to humans. DMSO have similar melting temperatures, G3TFSA will Furthermore, by using these models, we could look into return to a liquid state much quicker than DMSO. Our more reported effects known to be caused by these 10 mL stock of G3TFSA stored at − 20 °C took approxi- treatments. matively 10 min to melt at room temperature (around 20 ° Besides, in order to fully establish whether G3TFSA C) while it took slightly over 1 h for the 10 mL stock of and G4TFSA can be used as replacement organic sol- DMSO stored at − 20 °C to completely melt at room vents, the efficacy to deliver a wide range of compounds temperature making G3TFSA to return to a liquid state at and therapeutics needs to be assessed. The best way to least 6 time faster than DMSO. do this would be to use an established drug/compound Consequently, a study looking in to the effect of library which has an established and comprehensive freeze/thaw cycles on the stability of compounds in account of observed side effects and phenotypes. DMSO concluded that samples that underwent freeze/ thaw cycling suffered the most, showing a drop of more Conclusions than 10% in compound efficacy within 10 cycles [48], Our data reveals that both G3TFSA and G4TFSA are and after 25 freeze/thaw cycles tested, the percentage of comparable, at least, or slightly superior to DMSO in compound remaining was 55.8% [48]. Hence, since terms of compound deliverance as exhibited by the G4TFSA does not freeze at -20 °C, there is no risk of penetrance of DEAB and SU5402 (the latter only soluble compound degradation due to freeze/thaw cycles. in both DMSO and G4TFSA). This was evident by Moreover, SU5402 was used as a drug of choice in DEAB solubilized in G3TFSA and G4TFSA exhibiting evaluating the efficacy of the SILs as it is useful for the same phenotypic characteristics of DEAB made up assessing requirements for FGF signalling in the later in DMSO. SU5402 solubilized in G4TFSA and DMSO, stage of development of the zebrafish embryo because it reduced the expression of dlx2a in zebrafish embryos can be applied in late developmental events such as although to a greater extent for the latter. However, in organogenesis, leaving early FGF-dependent processes regards to the storage of DEAB stock solutions in unaffected. Additionally, SU5402 treatment potentially DMSO, G3TFSA and G4TFSA in the more conventional uncovers FGF requirements that are not revealed by manner at − 20 °C, G4TFSA remained in a liquid state knocking out specific FGF ligands or receptors owing to as it had a much lower glass-transition temperature, po- redundancy. tentially realising a decreased rate of sample degradation. In making up the stock solution of SU5402 in the Consequently, G3TFSA and G4TFSA solubilize and respective solvents; DMSO, G3TFSA and G4TFSA, it deliver test/pharmacological compounds adequately and was observed that SU5402 was not soluble in G3TFSA. routinely in research laboratories, hence both G3TFSA While the structure of both solvate ionic liquids is very and G4TFSA are suitable replacements for DMSO for similar, and they possess similar physical properties, the experimental procedures. poly-ether used to fabricate G4TFSA possesses an extra ethylene unit potentially increasing the solubilising Methods power of this liquid for small organic molecules [5]. Animal husbandry Hence, only SU5402 solubilized in G4TFSA was used to Zebrafish were reared and staged at 28.5 °C according to assess SU5402 phenotypic penetrance against SU5402 Kimmel et al. [54] After spawning, embryos were col- solubilized in DMSO. lected and raised in a petri dish in embryonic medium Yoganantharajah et al. BMC Biotechnology (2018) 18:32 Page 6 of 7 E3. As a standard, we raised zebrafish embryos in 30 ml Institute for Frontier Materials, Deakin University, 75 Pigdons Road, Geelong, VIC 3216, Australia. of E3 with 60 embryos per tube. Received: 30 March 2018 Accepted: 2 May 2018 Treatments N,N-Diethylaminobenzaldehyde (DEAB) (Sigma, MO, USA) and SU5402 (Sigma, MO, USA) were dissolved in References 1. Welton T. Chem Rev. 1999;99:2071–83. different solvents: DMSO, G3TFSA and G4TFSA at a 2. Beggs KM, Perus MD, Servinis L, O'Dell LA, Fox BL, Gengenbach TR, concentration of 10 mM. This concentration was chosen Henderson LC. RSC Adv. 2016;6:32480–3. based on our previous published data on the toxicity of 3. Altimari JM, Delaney JP, Servinis L, Squire JS, Thornton MT, Khosa SK, Long BM, Johnstone MD, Fleming CL, Pfeffer FM, Hickey SM, Wride MP, Ashton these solvents [29]. Stock solutions on 10 mM were kept TD, Fox BL, Byrne N, Henderson LC. 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Comparison of solvate ionic liquids and DMSO as an in vivo delivery and storage media for small molecular therapeutics

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Abstract

Background: Solvate ionic liquids (SILs) are a new class of ionic liquids that are equimolar solutions of lithium bistrifluoromethanesulfonimide in either triglyme or tetraglyme, referred to as G3LiTFSA and G4LiTFSA, respectively. SILs play a role in energy storage lithium batteries, and have been proposed as potential alternatives to traditional organic solvents such as DMSO. G3TFSA and G4TFSA have been shown to exhibit no toxicity in vivo up to 0.5% (v/ v), and solubilize small compounds (N,N-diethylaminobenzaldehyde) with full penetrance, similar to DMSO delivered DEAB. Herein, we compare the effects of storage (either at room temperature or − 20 °C) on DEAB solubilized in either DMSO, G3TFSA or G4TFSA to investigate compound degradation and efficacy. Results: The findings show that DEAB stored at room temperature (RT) for 4 months solubilized in either G3TFSA, G4TFSA or DMSO displayed no loss of penetrance. The same was observed with stock solutions stored at − 20 °C for 4 months; however G4TFSA remained in a liquid state compared to both G3TFSA and DMSO. Moreover, we examined the ability of G3TFSA and G4TFSA to solubilize another small molecular therapeutic, the FGFR antagonist SU5402. G4TFSA, unlike G3TFSA solubilized SU5402 and displayed similar phenotypic characteristics and reduced dlx2a expression as reported and shown with SU5402 in DMSO; albeit more penetrative. Conclusion: This study validates the use of these ionic liquids as a potential replacement for DMSO in vivo as organic solubilizing agents. Keywords: Zebrafish, Embryogenesis, Retinoic acid, Aldh1a2, FGFR, DEAB, SU5402, Ionic liquids Background pharmaceutical ingredients to ionic liquid-like salts, typic- In recent years, there has been an increase in the interest ally by inclusion of a charge diffuse cation or anion, which of ionic liquids (ILs) due to their potential in a myriad of can result in improved therapeutic effect via changes in chemical processes. Their unique property of being mol- crystal structure [18, 19]. These approaches have largely ten salts at room temperature imparts unusual properties revolved around the use of imidazolium-derived ionic liq- such as; high ionic conductivity, non-flammability, and uids which are a well-used and studied class of solvents. negligible vapour pressure. Due to these properties, and Of particular interest has been a new class of ionic their high customisability through anion/cation pairing, liquids, termed ‘solvate ionic liquids’ (SILs), reported by have become a staple material used throughout a variety Watanabe et al. [20–26]. The preparation of SILs is of disciplines [1–9]. The use of imidazolium ionic liquids trivial, being simple dissolution of LiNTf (lithium in drug delivery has seen some success, as they offer the bistrifluoromethanesulfonimide) in either triglyme potential to deliver sparingly soluble molecules via oral, or (triethylene glycol dimethyl ether, G3) or tetraglyme (tet- transdermal routes over a long period of time [10–17]. raethylene glycol dimethyl ether, G4) yields the ionic liq- Complementing this effect is the conversion of active uids, G3TFSA or G4TFSA, respectively (Fig. 1). Recent work by our group, and others, has characterised these * Correspondence: y.gibert@deakin.edu.au solvate ionic liquids using Kamlet-Taft parameters, and Metabolic Genetic Diseases Laboratory, Metabolic Research Unit, Deakin explored their use as a solvent for organic chemical University School of Medicine, 75 Pigdons Road, Geelong, VIC 3216, Australia transformations [27, 28]. Recently, we demonstrated the Full list of author information is available at the end of the article © The Author(s). 2018 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Yoganantharajah et al. BMC Biotechnology (2018) 18:32 Page 2 of 7 Fig. 1 Solvate ionic liquids and DMSO which are the focus of this study toxicity of solvate ionic liquids in vivo using a zebrafish G4TFSA, and their ability to solubilize and deliver other (Danio rerio), and found that both G3TFSA and small molecules/pharmacological compounds. G4TFSA with concentrations up to 50 μM (or 0.5%) are not toxic to zebrafish embryos (which are more sensitive to toxicity than adults) [29]. Since most organic modifier Results solvents (such as DMSO) are used at a much lower con- Evaluation of SILs stored at -20 °C centration (usually 0.1%), our study was able to conclude To be consistent with our previous study, which used the that both G3TFSA and G4TFSA can be safely used as retinoic acid synthesis inhibitor 4-diethylaminobenzaldehyde aqueous modifier solvents to allow evaluation of small (DEAB), we continued to use this molecule as the initial molecules. Both G3TFSA and G4TFSA do not induce focus of this work. DEAB is a known inhibitor of retinalde- apoptosis at a similar concentration to DMSO (10 μM) hyde dehydrogenases ALDH1A1, ALDH1A2, and and display a full drug penetrance and the anticipated ALDH1A3 in mammals (teleost fish do not have an aldh1a1 physiological changes induced in the test specimens gene) [34]. Retinaldehyde dehydrogenases convert retinalde- [29]. Since these novel SILs were able to replace DMSO hyde, a product from retinol (Vitamin A1), into retinoic acid as organic modifiers, we were curious if they could be (RA) [35]. Hence, inhibiting the function of retinaldehyde used as an alternative long term storage media for these dehydrogenases via DEAB abolishes the synthesis of RA molecules, and if compound degradation over this required for normal growth and development [36]. Thus, period was reduced compared to DMSO. we wanted to evaluate G3TFSA and G4TFSA’s ability to Currently, the most commonly used organic solvent, keep DEAB stable at RT without compound degradation or in academic and industrial research laboratories, to loss of efficacy. The rationale behind this test is that stock solubilize small organic molecules in water is DMSO solutions kept at RT would remain in a liquid state with no [30]. Due to its ability to dissolve many kinds of com- concern for sample degradation from multiple freeze/thaw pounds, DMSO plays a pivotal role in sample manage- cycles. This was evaluated over a duration of 4 months, and ment and high-throughput screening during in vivo and differences in penetrance of the drug, measured by the in vitro evaluation. strength of the phenotypes were determined. Due to its broad solubilizing capability and apparent To conduct this comparison, N,N-diethylaminobenzal- low toxicity at concentrations < 10%, [31, 32] DMSO is dehyde (DEAB), was administered in parallel to zebrafish used as a solvent for many drug types and is used as the embryos: one sample containing DMSO only (0.1%), one vehicle control of choice for both in vitro and in vivo DEAB in DMSO and the other two, containing DEAB in studies. However, in a study coordinated by Corderio either G3TFSA or G4TFSA. Zebrafish embryos were et al., the authors demonstrated low-dose toxicity of exposed to DEAB in the respective solvents at the begin- DMSO and concluded that solvents other than DMSO ning of gastrulation occurring at 6 h post fertilization should be employed for solubilizing drugs [33]. Thus (hpf) at a final concentration of 5 μM. there is a need to find a suitable replacement for DMSO Comparison of the treatments kept at RT showed that which do not possess this inherent toxicity. zebrafish embryos exposed to DEAB solubilised in either Therefore, the focus and aim of this study was to DMSO (Fig. 2b), G3TFSA (Fig. 2c), or G4TFSA (Fig. 2d) evaluate the performance of the ILs G3TFSA and from 6 to 30 hpf displayed characteristics associated G4TFSA as a potential replacement to the conventional with the loss of RA signalling compared to the control solvent DMSO, using zebrafish as a model organism. (Fig. 2a): [29, 37, 38] lack of pectoral fin induction Since, we have shown the ability of G3TFSA and (arrowhead), shortening of the posterior head (marked G4TFSA to solubilize DEAB with full penetrance of by an asterisk) malformation of the otic vesicle (arrow) reported phenotypic characteristics [29]; we wanted to and pericardiac oedema. There was no discernible differ- evaluate the impact of storage on both G3TFSA and ences in DEAB penetrance between DEAB in either Yoganantharajah et al. BMC Biotechnology (2018) 18:32 Page 3 of 7 embryos treated with 5 μM showed that zebrafish em- bryos exposed to DEAB solubilised in either DMSO (Fig. 3b), G3TFSA (Fig. 3c), or G4TFSA (Fig. 3d) from 6 to 30 hpf displayed the characteristics associated with the loss of RA signalling compared to the control (Fig. 3a)[29, 37, 38] (100% penetrance for all com- pounds tested, n =60). As previously observed in the RT test, there was an observed phenotype that clearly showed a lack of pec- toral fin induction (arrowhead), shortening of the poster- ior head (marked by an asterisk), malformation of the otic vesicle (arrow) and pericardiac oedema. However, unlike both DMSO and G3TFSA, G4TFSA did not freeze at − 20 °C but remained in a viscous state. Fig. 2 Room temperature DEAB exposure in developing zebrafish Capacity of the SILs to deliver small compounds embryos. Embryos exposed to DEAB at 5 μM in solution at room With these data in hand, our attention turned to demon- temperature for 4 months in either DMSO (b), G3TFSA (c), or G4TFSA (d) displayed the reported characteristic of loss of RA (compared to strating the generality of these ionic liquids as a storage control (DMSO exposed only (a): lack of pectoral fin induction and delivery media for small molecular therapeutics. For (arrowhead), shortening of the posterior head (asterisk), the purpose of this study, we evaluated the ability of malformation of the otic vesical (arrow) and pericardial edema both G3TFSA and G4TFSA to solubilise and compare the performance of SU5402 (Fig. 4d) a pan-fibroblast DMSO, G3TFSA or G4TFSA (100% for each, n = 60) growth factor receptors (FGFR) specific tyrosine kinase after storage at RT for 4 months [29]. inhibitor and is used in a multitude of zebrafish develop- mental systems to specifically inhibit FGFR signalling Evaluation of SILs stored at room temperature against DMSO [39–43]. A comparison of zebrafish em- To assess the solvate properties of G3TFSA and bryos treated with both SU5402 in G4TFSA at concen- G4TFSA, it was also imperative to evaluate the perform- trations 2.5 μM (Fig. 4c) and 5 μM (Fig. 4f) from 6 to 30 ance of the ionic liquids G3TFSA and G4TFSA after hpf displayed previously reported phenotypes, such as storage in the conventional (frozen) manner. Therefore, lack of pectoral fin induction (arrowhead) malformation DEAB was stored at − 20 °C for 4 months in either of the otic vesicle (arrow) compared to the control em- DMSO, G3TFSA or G4TFSA. A comparison of zebrafish bryo (Fig. 4a). Embryos treated with SU5402 in DMSO (Fig. 4b) also reported these phenotypes. There was also an evident lack of a formed mid-hindbrain boundary at concentrations of only 5 μM DEAB in DMSO and G4TFSA (open arrowhead) (Fig. 4e, f)[38, 44]. Assessing penetrance of the SILs In addition, we wanted to the examine genes that are affected by FGF (fibroblast growth factor) signalling. Hence, we investigated the effect on dlx2a expression using whole mount in situ hybridisation (WISH) after exposure to 2.5 μM SU5402 that had been either solubi- lized in DMSO or G4TFSA (Fig. 5). WISH results showed that compared to the control embryos (Fig. 5a, d), embryos treated with SU5402 solubilized in both DMSO (Fig. 5b, e) and G4TFSA (Fig. 5c, f) depicted an absence of dlx2a expression in the ventral cranial neural Fig. 3 Frozen DEAB exposure in developing zebrafish embryos. crest cells (arrows) marked by an asterix. However, Embryos exposed to DEAB at 5 μM that have been stored for comparatively there was a greater reduction in the 4 months at -20 °C in either DMSO (b), G3TFSA (c), or G4TFSA (d) display the reported characteristics of loss of RA (compared to expression of dlx2a (marked by AP staining) in the tel- control (DMSO exposed only (a): lack of pectoral fin induction encephalon (arrowhead) in zebrafish embryos that were (arrowhead), shortening of the posterior head (asterisk), treated with SU5402 solubilized in G4TFSA (Fig. 5c, f) malformation of the otic vesical (arrow) and pericardial edema compared to those treated with SU5402 solubilized in Yoganantharajah et al. BMC Biotechnology (2018) 18:32 Page 4 of 7 Fig. 4 SU5402 exposure in developing zebrafish embryos. Embryos are exposed to SU5402 (d)at2.5 μM(b & c)or5 μM(e & f) in either DMSO or G4TFSA display the reported characteristics of FGF signaling inhibition compared to control (untreated (a) or DMSO exposed only (b & e)): lack of pectoral fin induction (marked by an asterisk), malformation of the otic vesicle (arrow). Lloss of MHB (open arrow head) was observed in embryos treated with 5 μM DEAB. 100× magnification DMSO (Fig. 5b, e). This indicates that G4TFSA has as solutions in an environment of low relative humidity, to greater ability to deliver SU5402 (penetrative power) mitigate or retard compound degradation [45]. A study compared to DMSO. conducted by Kozikowski et al., investigating the effect of room temperature storage on the stability of com- Discussion pounds in DMSO concluded that the relationship be- Previous studies have shown that the prolonged storage tween length of storage and the probability of observing of organic compounds in solution can lead to significant the compound is described by a repeated measures sample degradation, and subsequently an increase in the logistic regression model [45]. Results from the study number of false positives for high-throughput biological determined that the probability of observing the com- screening assays [45]. These false positives represent po- pound was 92% after 3 months of storage at room tentially erroneous investment of time and money elab- temperature, 83% after 6 months, and 52% after 1 year orating on a false lead compound. As a result, many in DMSO [45]. Hence, it is valuable to assess the long researchers and pharmaceutical organizations now store term effects of RT storage on DEAB penetrance post their organic compounds as frozen DMSO stock solubilisation in both G3TFSA and GF4TFSA. Fig. 5 Expression of dlx2a visualized using whole mount in situ hybridization. Embryos exposed to SU5402 at 2.5 μM in either DMSO (b & e)or G4TFSA (c & f) display reduced localization and expression of dlx2a compared to control (DMSO exposed only (a & d) in the hindbrain (arrow) and in the pharyngeal arches (arrow head, * lack of pharyngeal arches). The first row depicts the embryos in a later orientation, the second row depicts the embryos in a ventral orientation Yoganantharajah et al. BMC Biotechnology (2018) 18:32 Page 5 of 7 Furthermore, a key characteristic of DMSO is that A previous study by Gibert et al., reported the forma- compared to G4TFSA it has a relatively higher melting tion of oral and pharyngeal dentition in teleosts depends point (T ) of around 19 °C [46], hence DMSO freezes on differential recruitment of retinoic acid signalling and easily and remelts slowly at room temperature. This that a lack of FGF signalling affects the expression of means that if stored frozen, a considerable amount of dlx2a (distal-less homeobox 2a) [49]. Dlx genes are time will be spent getting DMSO (and the compound expressed in a coordinate manner which create proximal solubilized in DMSO) to a liquid state before it can be to distal polarity within the pharyngeal arches [50]. In zeb- used. Whereas, G4TFSA was able to remain in a liquid rafish, dlx2a is expressed in the migrating cranial neural like state at − 20 °C, proving advantageous over DMSO which contributes to the pharyngeal arches [49–53]. as it can be used straight away after being removed from In order to get a more accurate understanding into storage at − 20 °C. Similar to DMSO, G3TFSA albeit whether these SILs can be used clinically we will need to higher, has a similar T of 23 °C [47]. However, G3TFSA continue our studies in adult and mammalian models. has a much higher entropy change of fusion value of Even though zebrafish provide an adequate starting − 1 − 1 112.5 J K mol compared to DMSO which only has an point, the efficacy and penetrance of G3TFSA and − 1 − 1 entropy change of fusion value of around 50 J K mol G4TFSA would need to be assessed in a mammalian [47]. This means that even though both G3TFSA and model which would share more homology to humans. DMSO have similar melting temperatures, G3TFSA will Furthermore, by using these models, we could look into return to a liquid state much quicker than DMSO. Our more reported effects known to be caused by these 10 mL stock of G3TFSA stored at − 20 °C took approxi- treatments. matively 10 min to melt at room temperature (around 20 ° Besides, in order to fully establish whether G3TFSA C) while it took slightly over 1 h for the 10 mL stock of and G4TFSA can be used as replacement organic sol- DMSO stored at − 20 °C to completely melt at room vents, the efficacy to deliver a wide range of compounds temperature making G3TFSA to return to a liquid state at and therapeutics needs to be assessed. The best way to least 6 time faster than DMSO. do this would be to use an established drug/compound Consequently, a study looking in to the effect of library which has an established and comprehensive freeze/thaw cycles on the stability of compounds in account of observed side effects and phenotypes. DMSO concluded that samples that underwent freeze/ thaw cycling suffered the most, showing a drop of more Conclusions than 10% in compound efficacy within 10 cycles [48], Our data reveals that both G3TFSA and G4TFSA are and after 25 freeze/thaw cycles tested, the percentage of comparable, at least, or slightly superior to DMSO in compound remaining was 55.8% [48]. Hence, since terms of compound deliverance as exhibited by the G4TFSA does not freeze at -20 °C, there is no risk of penetrance of DEAB and SU5402 (the latter only soluble compound degradation due to freeze/thaw cycles. in both DMSO and G4TFSA). This was evident by Moreover, SU5402 was used as a drug of choice in DEAB solubilized in G3TFSA and G4TFSA exhibiting evaluating the efficacy of the SILs as it is useful for the same phenotypic characteristics of DEAB made up assessing requirements for FGF signalling in the later in DMSO. SU5402 solubilized in G4TFSA and DMSO, stage of development of the zebrafish embryo because it reduced the expression of dlx2a in zebrafish embryos can be applied in late developmental events such as although to a greater extent for the latter. However, in organogenesis, leaving early FGF-dependent processes regards to the storage of DEAB stock solutions in unaffected. Additionally, SU5402 treatment potentially DMSO, G3TFSA and G4TFSA in the more conventional uncovers FGF requirements that are not revealed by manner at − 20 °C, G4TFSA remained in a liquid state knocking out specific FGF ligands or receptors owing to as it had a much lower glass-transition temperature, po- redundancy. tentially realising a decreased rate of sample degradation. In making up the stock solution of SU5402 in the Consequently, G3TFSA and G4TFSA solubilize and respective solvents; DMSO, G3TFSA and G4TFSA, it deliver test/pharmacological compounds adequately and was observed that SU5402 was not soluble in G3TFSA. routinely in research laboratories, hence both G3TFSA While the structure of both solvate ionic liquids is very and G4TFSA are suitable replacements for DMSO for similar, and they possess similar physical properties, the experimental procedures. poly-ether used to fabricate G4TFSA possesses an extra ethylene unit potentially increasing the solubilising Methods power of this liquid for small organic molecules [5]. Animal husbandry Hence, only SU5402 solubilized in G4TFSA was used to Zebrafish were reared and staged at 28.5 °C according to assess SU5402 phenotypic penetrance against SU5402 Kimmel et al. [54] After spawning, embryos were col- solubilized in DMSO. lected and raised in a petri dish in embryonic medium Yoganantharajah et al. BMC Biotechnology (2018) 18:32 Page 6 of 7 E3. As a standard, we raised zebrafish embryos in 30 ml Institute for Frontier Materials, Deakin University, 75 Pigdons Road, Geelong, VIC 3216, Australia. of E3 with 60 embryos per tube. Received: 30 March 2018 Accepted: 2 May 2018 Treatments N,N-Diethylaminobenzaldehyde (DEAB) (Sigma, MO, USA) and SU5402 (Sigma, MO, USA) were dissolved in References 1. Welton T. Chem Rev. 1999;99:2071–83. different solvents: DMSO, G3TFSA and G4TFSA at a 2. Beggs KM, Perus MD, Servinis L, O'Dell LA, Fox BL, Gengenbach TR, concentration of 10 mM. This concentration was chosen Henderson LC. RSC Adv. 2016;6:32480–3. based on our previous published data on the toxicity of 3. Altimari JM, Delaney JP, Servinis L, Squire JS, Thornton MT, Khosa SK, Long BM, Johnstone MD, Fleming CL, Pfeffer FM, Hickey SM, Wride MP, Ashton these solvents [29]. Stock solutions on 10 mM were kept TD, Fox BL, Byrne N, Henderson LC. 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BMC BiotechnologySpringer Journals

Published: May 29, 2018

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