Journal Pre-proof Ionic liquids as protein stabilizers for biological and biomedical applications: A review Nathalia Vieira Veríssimo, Filipa A. Vicente, Rodrigo Cardoso de Oliveira, Blaž Likozar, Ricardo Pereira de Souza Oliveira, Jorge Fernando Brandão Pereira PII: S0734-9750(22)00151-3 DOI: https://doi.org/10.1016/j.biotechadv.2022.108055 Reference: JBA 108055 To appear in: Biotechnology Advances Received date: 5 August 2022 Revised date: 13 October 2022 Accepted date: 23 October 2022 Please cite this article as: N.V. Veríssimo, F.A. Vicente, R.C. de Oliveira, et al., Ionic liquids as protein stabilizers for biological and biomedical applications: A review, Biotechnology Advances (2022), https://doi.org/10.1016/j.biotechadv.2022.108055 This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2022 Published by Elsevier Inc. Journal Pre-proof Review Ionic liquids as protein stabilizers for biological and biomedical applications: A review Nathalia Vieira Veríssimo 1, Filipa A. Vicente 2, Rodrigo Cardoso de Oliveira 1, Blaž Likozar 2, Ricardo Pereira de Souza Oliveira,1 and Jorge Fernando Brandão Pereira 3 * 1 f School of Pharmaceutical Sciences, São Paulo University (USP), Av. Prof. Lineu Prestes, no. 580, B16, 05508-000, Cidade de Universitária, São Paulo, SP, Brazil. N. V. Veríssimo: nathaliavieira@usp.br; R. C. Oliveira: rc.oliveira@usp.br; R. P S. Oliveira: rpsolive@usp.br. 2 oo Department of Catalysis and Chemical Reaction Engineering, National Institute of Chemistry, Hajdrihova 19, 1000 Ljubljana, Slovenia. F. A. Vicente: filipa.andre.vicente@ki.si; B. Likozar: Blaz.Likozar@ki.si. 3 e- pr Univ Coimbra, CIEPQPF, Department of Chemical Engineering, Rua Sílvio Lima, Pólo II – Pinhal de Marrocos, 3030-790 Coimbra, Portugal. Jo u rn al Pr * Corresponding author: Univ Coimbra, CIEPQPF, Department of Chemical Engineering, Rua Sílvio Lima, Pólo II – Pinhal de Marrocos, 3030-790 Coimbra, Portugal; jfbpereira@eq.uc.pt; Tel: + 351 239 798 726. 1 Journal Pre-proof Abstract pr oo f Biotechnology has revolutionized science and health care by providing new biomolecules with biological and medical applications. However, the low stability of several life-saving bioproducts still hinders their transport, storage, and application. Hence, protein-based bioproducts instability and high costs are the main bottlenecks limiting access to biopharmaceuticals in low-income countries and communities. Aiming to improve the stability of protein-based products, researchers have studied ionic liquids (ILs) as protein stabilizers due to their unique properties and ability to enhance the solubility and stability of a wide range of biomolecules. Although different classes of ILs have the potential to improve protein stability, their effects are dependent on several variables, such as the complex and intrinsic properties of proteins, the nature and concentration of ILs, and environmental conditions (e.g., temperature, pH). For medical applications, the biocompatibility of ILs can also limit their biological use. Therefore, the current state-of-the-art on ILs applications for non-enzymatic protein stabilization was carefully analyzed and discussed, considering protein properties, ILs classes, and IL solutions concentrations. Lastly, a critical perspective regarding ILs applications as protein stabilizers was presented, highlighting the current lacunas in the field while guiding future studies to answer the existing paradigms. Jo u rn al Pr e- Keywords: protein stability; preservatives; ionic liquids; biopharmaceuticals; pharmaceutical formulations. 2 Journal Pre-proof Index 1. Introduction .............................................................................................................. 9 2. Protein structure and stability .................................................................................... 9 2.1. Determination of protein stability ..................................................................... 10 2.2. Protein stabilization .......................................................................................... 12 3. Ionic liquids for protein stabilization ....................................................................... 13 3.1. Interactions and effects ..................................................................................... 13 3.2. Effect of ILs on non-enzymatic proteins ........................................................... 15 3.2.1. Effect of ILs on non-enzymatic lipophilic and amphipathic proteins .......... 15 oo f 3.2.1.1. Insulin ................................................................................................. 18 3.2.1.2. Hemoglobin (Hb) ................................................................................ 20 3.2.2. Effect of ILs on non-enzymatic hydrophilic proteins .................................. 22 pr 3.2.2.1. Cytochrome C (Cyt C) ........................................................................ 29 e- 3.2.2.2. Green fluorescent proteins (GFP) ........................................................ 32 3.2.2.3. Myoglobin (Mb) ................................................................................. 34 Pr 3.2.2.4. Serum albumin .................................................................................... 36 3.2.2.4.1. Bovine serum albumin (BSA)....................................................... 36 al 3.2.2.4.2. Human serum albumin (HSA) ...................................................... 39 3.2.2.5. β-Lactoglobulin (BLG) ....................................................................... 41 rn 4. Final Remarks ......................................................................................................... 42 Jo u 4.1. Biocompatibility of ILs .................................................................................... 43 4.2. Perspective on the use ILs for the stabilization of protein-based bioproducts .... 44 4.3. SWOT analysis ................................................................................................ 50 3 Journal Pre-proof Abbreviations Proteins BLG, β-lactoglobulin (BLG) BSA, bovine serum albumin (BSA) Cyt C, cytochrome C (Cyt C) EGFP, enhanced green fluorescent protein (EGFP) GFP, green fluorescent protein (GFP) Hb, hemoglobin (Hb) HEWL, hen egg-white lysozyme (HEWL) Mb, myoglobin (Mb) SA, serum albumin (SA) oo immunoglobulin (IgG) pr IgG, f HSA, human serum albumin (HSA) sfGFP, superfolder green fluorescent protein (sfGFP) wild-type green fluorescent protein (wtGFP) e- wtGFP, Pr Ionic liquids Ammonium-based ILs al [N0,1,1,(2OH)][C2CO2], N,N-dimethylethanolamine propionate [N0,0,0,4]NO3, butylammonium nitrate rn [N0,0,2,2][CH₃COO], diethylammonium acetate [N0,0,2,2]PO₄, diethylammonium phosphate Jo u [N0,0,2,2]SO₄, diethylammonium sulfate [N0,0,0,2][CHO2], ethylammonium formate [N0,0,0,2][CH₃SO₃], ethylammonium methanesulfonate [N0,0,0,2]NO3, ethylammonium nitrate [N0,0,0,(2OH)]NO3, ethanolammonium nitrate [N0,0,0,1][CHO2], methylammonium formate [N4,4,4,4]OH, tetrabutylammonium hydroxide [N0,2,2,2][CH₃COO], triethylammonium acetate [N2,2,2,2]OH, tetraethylammonium hydroxide [N0,2,2,2][CH₃SO₃], triethylammonium methanesulfonate [N0,2,2,2]PO₄, triethylammonium phosphate [N0,2,2,2]SO₄, triethylammonium sulfate [N0,2,2,2][CF₃COO], triethylammonium trifluoroacetate 4 Journal Pre-proof [N0,1,1,1][CH₃COO], trimethylammonium acetate [N1,1,1,1]OH, tetramethylammonium hydroxide [N0,1,1,1]H2PO4, trimethylammonium dihydrogen phosphate [N0,1,1,1]HSO4, trimethylammonium hydrogen sulfate [N3,3,3,3]OH, tetrapropylammonium hydroxide Cholinium-based ILs [Ch][(CH3(CH2)3)2HPO4], cholinium dibutylphosphate [Ch][Arg], cholinium L-arginate [Ch][Asn], cholinium L-asparaginate oo f [Ch][CH₃COO], cholinium acetate [Ch][CH₃SO₃], cholinium methanesulfonate pr [Ch][Gln], cholinium L-glutaminate [Ch]₂[Asn], dicholinium L-asparaginate [Ch]₂[Gln], dicholinium L-glutaminate e- [Ch][Lys], cholinium L-lysinate Pr [Ch]H2PO4, cholinium dihydrogen phosphate [diHOHTMGu]Cl, al Guanidinium-based ILs N,N,N,N-tetramethyl-N,N-hexanol-guanidinium chloride tetramethylguanidinium methylacetate Jo u rn [TMGu][CH₃CH₂COOH], Imidazolium-based ILs [AMIm]Cl, 1-allyl-3-methylimidazolium chloride [C₁₀MIm][CH₃COO], 1-decyl-3-methylimidazolium acetate [C₁₀MIm]Cl, 1-decyl-3-methylimidazolium chloride [C₁₂MIm][CH₃COO], 1-dodecyl-3-methylimidazolium acetate [C₁₂MIm]Cl, 1-dodecyl-3-methylimidazolium chloride [C₁₄MIm]Br, 1-tetradecyl-3-methylimidazolium bromide [C₁C1Im]Cl, 1,3-dimethylimidazolium chloride [C₂MIm][CH₃COO], 1-ethyl-3-methylimidazolium acetate [C₂MIm][CH₃SO₃], 1-ethyl-3-methylimidazolium methanesulfonate [C₂MIm][EtSO₄], 1-ethyl-3-methyl imidazolium ethylsulfate [C₂MIm][Me₂PO₄], 1-ethyl-3-methylimidazolium dimethylphosphate [C₂MIm][N(CN)2], 1-ethyl-3-methyl imidazolium dicyanamide 5 Journal Pre-proof [C₂MIm][Phe], 1-ethyl-3-methylimidazolium phenylalanine [C₂MIm]BF₄, 1-ethyl-3-methylimidazolium tetrafluoroborate [C₂MIm]Cl, 1-ethyl-3-methylimidazolium chloride [C₂MIm]NO₃, 1-ethyl-3-methyl imidazolium nitrate [C₂MIm]SCN, 1-ethyl-3-methylimidazolium thiocyanate [C₂MIm][Tf₂N], 1-ethyl-3-methylimidazolium bis(trifluoromethylsulfonyl)imide [C₂OCMIm]Cl, 1-(2-methoxyethyl)-3-methylimidazolium chloride [C4C4Im]Cl, 1,3-dibutylimidazolium chloride [C₄C1C1Im]Cl, 1-butyl-2,3-dimethylimidazolium chloride 1-ethyl-3-methylimidazolium tricyanomethanide [C₄MIm][C₈SO₄], 1-butyl-3-methylimidazolium octyl sulfate [C₄MIm][C₈SO₄], 1-butyl-3-methylimidazolium octyl sulfate oo f [C₄MIm][C(CN)₃], pr [C₄MIm][CF₃COO], 1-butyl-3-methylimidazolium trifluoroacetate [C₄MIm][CH₃COO], 1-butyl-3-methylimidazolium acetate 1-butyl-3-methylimidazolium methanesulfonate [C₄MIm][Lac], 1-butyl-3-methylimidazolium lactate [C₄MIm][MeSO₄], 1-butyl-3-methylimidazolium methylsulfate [C₄MIm][N(CN)2], 1-butyl-3-methylimidazolium dicyanamide Pr e- [C₄MIm][CH₃SO₃], al [C₄MIm]BF₄, 1-butyl-2,3-dimethylimidazolium tetrafluoroborate 1-butyl-3-methylimidazolium bromide [C₄MIm]Cl, 1-butyl-3-methylimidazolium chloride rn [C₄MIm]Br, [C₄MIm]HSO₄, 1-butyl-3-methylimidazolium hydrogensulfate Jo u [C₄MIm]I, 1-butyl-3-methylimidazolium iodine [C₄MIm]NO₃, 1-butyl-3-methylimidazolium nitrate [C₄MIm]PF₆, 1-butyl-3-methylimidazolium hexafluorophosphate [C₄MIm]SNC, 1-ethyl-3-methylimidazolium thiocyanate [C₄MIm][Tf₂N], 1-butyl-3-methylimidazolium bis(trifluoromethylsulfonyl)imide [C₄MIm][TfO], 1-butyl-3-methylimidazolium trifluoromethanesulfonate [C₅MIm]Br, 1-pentyl-3-methylimidazolium bromide [C₆MIm][C₁₀SO₄], 1-hexyl-3-methylimidazolium dodecyl sulfate [C₆MIm][CH₃COO], 1-hexyl-3-methylimidazolium acetate [C₆MIm]BF₄, 1-hexyl-3-methylimidazolium tetrafluoroborate [C₆MIm]Br, 1-hexyl-3-methylimidazolium bromide [C₆MIm]Cl, 1-hexyl-3-methylimidazolium chloride [C₈MIm][CH₃COO], 1-octyl-3-methylimidazolium acetate 6 Journal Pre-proof [C₈MIm]Br, 1-octyl-3-methylimidazolium bromide [C₈MIm]Cl, 1-octyl-3-methylimidazolium chloride [EtOCOCH₂MIm][C₁₀SO₄], 3-methyl-1-(ethoxycarbonylmethyl)imidazolium dodecyl sulfate [EtOCOCH₂MIm][C₁₀SO₄], 3-methyl-1-(ethoxycarbonylmethyl)imidazolium dodecyl sulfate [OHC2MIm]Cl, 1-(2-hydroxyethyl)-3-methylimidazolium chloride Phosphonium-based ILs f [P4,4,4,4][SS], tetrabutylphosphonium styrenesulfonate oo Pyridinium- and pyrrolidinium-based ILs [C4C1OPyrr]Br, 1-butyl-1-methyl-2-oxopyrrolidinium bromide pr [C4C1Pyrr][CH₃COO], 1-butyl-4-methyl pyrrolidinium acetate [C4C1Pyrr][TfO], 1-butyl-1-methylpyrrolidinium trifluoromethanesulfonate e- [C4C1Pyrr][Cl, 1-butyl-4-methyl pyrrolidinium chloride Pr [C4C1Pyr]H2PO4, 1-butyl-1-methylpyridinium dihydrogen phosphate [EtOCOCH₂Pyrr][C₁₀SO₄], 3-methyl-1-(ethoxycarbonylmethyl)pyrrolidinium dodecyl sulfate rn al [EtOCOCH₂Pyrr][C₁₀SO₄], 3-methyl-1-(ethoxycarbonylmethyl)pyrrolidinium dodecyl sulfate Other variations in Gibbs free energy Jo u ΔG, ∆Hm, melting enthalpy ABS, aqueous biphasic systems ATP, adenosine triphosphate CD, circular dichroism CMC, critical micelle concentration Cryo-EM, cryogenic electron microscopy DSC, differential scanning calorimetry DSF, differential scanning fluorimetry EMA, European Medicines Agency FDA, Food and Drug Administration FTIR, Fourier transform infrared GRAVY, average hydrophobicity index 7 Journal Pre-proof GuHCl, guanidinium hydrochloride H₂O₂, hydrogen peroxide II, instability index ILs, ionic liquids ITC, isothermal titration calorimetry K₂HPO₄, dipotassium hydrogen phosphate KH₂PO₄, potassium dihydrogen phosphate M, molar MD, molecular dynamics f differential scanning micro-calorimetry MW, molecular weight NaCl, sodium chloride pr NMR, nuclear magnetic resonance spectroscopies isoelectric point SAILs, surface-active ionic liquids Pr SANS, small-angle neutron scattering e- PBS, with sodium phosphate buffer pI, oo micro-DSC, SAXS, small-angle X-ray scattering melting temperature UN SDGs, United Nations Sustainable Development Goals rn Tm, al SDS, sodium dodecyl sulfate Jo u WHO, World Health Organization 8 Journal Pre-proof 1. Introduction Over the last decades, advances in biotechnology have revolutionized health care, leading to the approval of over 260 novel human therapeutics (Evens and Kaitin, 2015). This allowed the treatment and increase in the survival rate of patients with conditions previously considered “incurable”, such as certain types of cancer and autoimmune diseases (Walsh, 2013). Nevertheless, despite their great potential, proteinbased biotechnological products are still inaccessible to many disadvantaged communities and low-income countries (Ferrari, 2022; Oliveira et al., 2015). This is mostly due to their low stability either in liquid formulations or upon lyophilization, which hinders their distribution, storage, and application (Manning et al., 2010). Therefore, stabilizers are often added to prevent the unfolding and aggregation of these proteins during manufacture, transport, and long-term storage, or to protect the proteins against the physical stress associated with the lyophilization process. rn al Pr e- pr oo f Common stabilizers and excipients added include sugars, polyols, amino acids, polymers, surfactants, kosmotropic salts, and osmolytes (naturally-occurring organic small molecules) (Butreddy et al., 2021; Bye et al., 2014). However, these are not transversal to stabilize all therapeutic proteins nor for their storage, representing a major bottleneck for biopharmaceutical stabilization. In this sense, the development of formulations that allow the preservation of protein-based bioproducts not only can facilitate their storage and transport but also expand their application, hence contributing to universal access to affordable and safe biotechnological products (Ferrari, 2022; Oliveira et al., 2015). Besides, this would also be contributing to goal 3 of the United Nations Sustainable Development Goals (UN SDGs) - Ensure healthy lives and promote well-being for all at all ages (UN – United Nations, 2022) - while representing an important step moving forward. To help solve these issues, different research groups have been demonstrating the potential of ionic liquids (ILs) as solvents or additives for protein stabilization (Kumar et al., 2017; Weingärtner et al., 2012) due to their enhanced ability to stabilize biomolecules like proteins and enzymes. Jo u With this in mind, this review compiles and discusses the current state-of-the-art on ILs applications for protein stabilization, but goes further than simply taking into account the different IL classes and the general protein properties. Herein, we used the UniProt sequence of the most usual variant of each type of protein to calculate its grand average hydrophobicity index, molecular weight, and instability index using the Expasy ProtParam tool (Artimo et al., 2012; ExPASy, 2018; UniProt, 2020). Hence, a more indepth analysis was provided alongside the different trends observed. Furthermore, this article presents and debates the compatibility of ILs with distinct biological systems. Lastly, a critical perspective is also provided based on a strengths, weaknesses, opportunities, and threats (SWOT) analysis in using ILs as proteins stabilizers, especially considering the lack of approved legislation by competent agencies, for instance, the European Medicines Agency (EMA) and the Food and Drug Administration (FDA). 2. Protein structure and stability Proteins are remarkably sophisticated macromolecular biological structures that, in addition to their complex organization, are flexible and can be easily rearranged according to the conditions of the surrounding environment (Mongan and Case, 2005). Although there are thousands of studies on protein composition, arrangement, and behavior, scientists are still scratching the surface of this topic. There are more than 340 million proteins registered in the non-redundant database of UniProt (UniParc) 9 Journal Pre-proof (UniProt, 2020) and 20,000 different proteins in the human body alone (Ponomarenko et al., 2016). Impressively, this estimate only considers unique proteins, since a single yeast cell can contain 42 million of them (Ho et al., 2018). In addition to its great diversity, every protein is also an intricate system itself. The structure of a protein is composed of a chain of amino acids in a three-dimensional arrangement, divided into four organizational levels (Nelson and Cox, 2012). Below, Fig. 1 depicts each of the different levels of protein structures. oo f Fig. 1. A) Primary structure of proteins – amino acid chain. B) Secondary structure of proteins – interactions of polypeptide chains: α-helix, β-sheet, and random coil. C) The tertiary structure of proteins – three-dimensional folding of the protein structure (demonstrated by the structure of the wild-type Green Fluorescent Protein, PDB ID: 1GFL). D) Quaternary structure of proteins – packing of different subunits of protein (demonstrated by the Human hemoglobin A, PDB ID: 1MKO). Images of the proteins were produced with the PDB structures using UCSF Chimera 1.14 (Berman et al., 2002; Pettersen et al., 2004). Jo u rn al Pr e- pr The primary structure is the amino acid chain [Fig. 1.A], the secondary comprehends the interactions of polypeptide chains (e.g., α-helix, β-sheets, coils) [Fig. 1.B], the tertiary corresponds to the overall three-dimensional folding of the protein structure [Fig. 1.C], and the quaternary describes the packing of different subunits of proteins formed by multiple polypeptide chains [Fig. 1.D] (Nelson and Cox, 2012). The arrangement of the protein structure is not static, and a plethora of conditions [e.g., pH, temperature, pressure, ionic strength, molecular interactions, and presence of chemical compounds] can reversibly or irreversibly alter them (Manning et al., 2010). Considering that the biological activity of proteins is intrinsically dependent on the integrity of their structure, a deeper understanding of the properties, structure, behavior, and stability of proteins is essential for their effective and safe use (Manning et al., 2010). Notably, stability and enhancement studies are fundamental to develop biomolecules-based applications and improve their industrial manufacturing, transport, and handling on a large scale. Hence, the next subsection presents the current approaches being use to determine proteins stability. 2.1. Determination of protein stability Multiple parameters can be used to evaluate protein stability. For example, altering the tertiary structure of a protein can impact its thermal stability but preserve its activity. In other cases, changes in the surface of the protein do not alter its tertiary structure but can decrease its biological activity (Fujita et al., 2007). Hence, the selection of adequate parameters and techniques to determine proteins stability varies according to the biomolecule and its intended application. With this in mind, this subsection will briefly discuss the main approaches to evaluate protein stability. As aforementioned, proteins present an intricate three-dimensional arrangement that goes from primary to quaternary structure (Nelson and Cox, 2012). The primary structure is usually resistant to stress, and only severe conditions or enzymes can break its peptide bonds (Bischof and He, 2006). To determine changes in the protein's amino acids or their sequence, it is possible to directly sequence the protein or evaluate its size using electrophoresis, eastern and western blotting, or chromatography (Deller et al., 2016). Furthermore, changes in the half-life of the protein can also be indicative of alterations in its primary structure (Deller et al., 2016). The most common method for protein sequencing is mass spectrometry (usually combined with chromatography techniques) (Callahan et al., 2020), but Edman degradation is still useful to characterize 10 Journal Pre-proof the N-terminus of proteins (Zhou et al., 2012). To evaluate alterations in the secondary structure of proteins, the most used methods include circular dichroism (CD) and infrared spectroscopies [e.g., Fourier transform infrared (FTIR), 2D-infrared] (Greenfield, 2006; Kong and Yu, 2007). al Pr e- pr oo f For the tertiary structure, the technique must show the folding and conformation of the protein or indicate changes to its three-dimensional arrangement. Direct methods that show the protein’s tertiary structure include X-ray crystallography, neutron and Xray scatterings [e.g., small-angle neutron scattering (SANS), small-angle X-ray scattering (SAXS)], nuclear magnetic resonance (NMR) spectroscopy, dual polarisation interferometry, and cryogenic electron microscopy (Cryo-EM) (Alberts et al., 2002; Ilari and Savino, 2008; Kikhney and Svergun, 2015; Milne et al., 2013; Petoukhov and Svergun, 2007; Swann et al., 2004) Additionally, there are also indirect approaches to evaluate changes in the tertiary structure of proteins. For instance, changes in fluorescence or absorbance of proteins with fluorescent amino-acid residues (i.e., tyrosine, tryptophan, and phenylalanine), with fluorophores or chromophores in its structure (e.g., fluorescent proteins), or with the addition of fluorophores to its structure (e.g., fluorescein conjugate) can indicate alterations to its tertiary structure (dos Santos et al., 2020, 2019). Furthermore, there are also computational tools to predict protein structure based on homology modeling (i.e., deducing a tertiary structure based on a homologous protein with known conformation), threading or fold recognition (i.e., predicting a tertiary structure based on proteins of similar sequence, when no homologous protein is available), and Ab initio structure prediction (i.e., deduces the tertiary structure based on the primary amino-acid sequence) (Lee et al., 2017; Watson et al., 2005; Xiang, 2006; Xu et al., 2008). Usually, more than one computational and experimental method is employed to determine protein tertiary structure, as all of them have their limitations and technique artifacts (Liu and Hsu, 2005; Watson et al., 2005). Jo u rn For the quaternary structure, the method must search for changes in the oligomeric state of the protein, which can be done with techniques such as size exclusion chromatography and electrophoresis (Deller et al., 2016) or the previously discussed strategies to determine protein tertiary structure. Moreover, these techniques can also be used to monitor the aggregation of proteins, which can be associated with loss of activity and denaturation of biomolecules (Wang, 2005; Wang et al., 2010). Assessing the thermal stability of proteins provides information not only regarding their resistance to heat, such as their melting temperature (Tm, i.e., temperature of denaturation), but it can also be used to estimate protein thermodynamic parameters, namely the melting enthalpy (∆Hm) and Gibbs Free Energy (ΔG) (Bischof and He, 2006; Hong et al., 2009). The Tm of proteins can be measured directly by differential scanning fluorimetry (DSF), isothermal titration calorimetry (ITC), and differential scanning calorimetry (DSC) (Bischof and He, 2006; Deller et al., 2016). Nevertheless, it is also possible to estimate T m by heating samples and monitoring alteration to its secondary structure using CD or FTIR. Finally, the evaluation of the protein’s biological activity depends on its intended application. For example, it can include kinetic parameters for enzymes, absorbance and fluorescence for biosensors, and binding, immunogenicity and immunomodulation tests for vaccines (dos Santos et al., 2020; Iyer and Ananthanarayan, 2008; Schofield, 2009). For other classes of biopharmaceuticals, it varies according to their application, as there is a vast range of pharmacological classes that require different assessments (Manning et al., 2010; Patel et al., 2011). 11 Journal Pre-proof After establishing the parameters and methods to evaluate the protein stability, the next subsection will discuss the main conditions that cause protein denaturation and the problems associated with protein instability. 2.2. Protein stabilization al Pr e- pr oo f The instability/low stability of most proteins in unfavorable environments, such as high temperatures, alkaline or acidic conditions, and the presence of denaturing compounds in solution (Manning et al., 2010, 1989) has limited the number of commercial applications of protein-based products. Finding solvents, additives, or preservatives able to maintain or improve the stability of proteins would allow the design of novel applications for bioproducts that contain them. Such findings would revolutionize the transportation and storage of protein-based products, like vaccines and insulin. For example, cities and rural settlements in low-income countries or peripheral communities with limited access to the electrical grid do not have the minimal conditions to maintain the refrigeration (generally close to or below 0 ºC) of proteinbased products during their distribution and storage (Humphreys, 2011; Zaffran et al., 2013). Consequently, these communities have limited access to essential biopharmaceuticals, generating critical Public Health concerns. According to the World Health Organization (WHO), vaccines save 2 to 3 million lives every year, yet vaccination coverage expansion could save an extra 1.5 million (WHO, 2020). Improving the stability of (bio)formulations could help ease this matter. For instance, more resistant (bio)formulations would generate lower losses during processing and have less strict transport and storage requirements (Manning et al., 2010, 1989). Therefore, discovering substances to preserve protein activity outside the cold-chain facilities would aid their distribution and expand access to biological products. Fig. 2 summarizes the main bottlenecks for protein application while also presenting potential solutions. Jo u rn Fig. 2. Schematic representation of the main issues and potential solutions for expanding the large-scale access to proteins of commercial interest. As shown in Fig. 2, increasing protein stability can positively impact the commercial application of proteins, allowing the development of novel uses and helping to solve current problems. It is possible to minimize losses during the industrial manufacture of proteins by increasing protein stability however, the improvement in stability is generally limited due to the unstable nature of proteins. Therefore, the use of excipients is common during the entire manufacturing process and in the final formulation to achieve protein stabilization through retardation of chemical degradation processes and prevention of aggregation. Protein stability is a result of achieving a balance between destabilizing and stabilizing forces. The destabilizing forces are mainly due to the large increase in entropy of unfolding, whereas the stabilizing forces are provided by intra-protein and protein-solvent interactions. Most common excipients promote this stabilization by their interaction with the protein, the container surface, and most importantly with water. When in solution, these excipients stabilize proteins by direct binding while others promote the formation of a hydration shell around the proteins, preventing the unfolding and further aggregation. Once the protein is lyophilized, the main stabilization effect results from the direct binding of the excipient with the protein (Ohtake et al., 2011). 12 Journal Pre-proof Among the different classes of stabilizers, some ILs have proven quite efficient in structuring the water shell around the proteins whereas others have connected to the proteins during the unfolding provoked by external stresses, which prevented the protein aggregation and later favored the refolding (Reslan and Kayser, 2018). Therefore, this will be explored in detail in the next section. 3. Ionic liquids for protein stabilization e- pr oo f Among the solvents with the potential to stabilize protein-based products, ILs emerge as promising candidates, especially considering that some have appropriate characteristics for biological and biomedical applications (Kunz and Häckl, 2016). ILs are compounds composed solely of nonsymmetric ions with low lattice energy and hence low melting points (Tan et al., 2012). Due to their ionic nature, they have outstanding properties. For instance, ILs are easily tailorable by altering the cationanion pair and can be designed to present suitable properties for pharmaceutical applications, such as biocompatibility with cells and biodegradability, high thermal stability, a capacity to solvate a wide range of compounds, and water solubility (Freire, 2016; Kunz and Häckl, 2016). In addition, and of course, always depending on their intrinsic chemical structure, ILs have also other useful characteristics for industrial processing, such as low viscosity, negligible vapor pressure, high thermal and chemical stability, and electrical conductivity. Several researchers have successfully explored and demonstrated the potential of ILs as protein stabilizers or activity enhancers (Kumar et al., 2017; Patel et al., 2011; Veríssimo et al., 2021). Jo u rn al Pr One of the pioneering studies was published in 2000 by Summers and Flowers (Summers and Flowers, 2000), which showed that the IL ethylammonium nitrate ([N0,0,0,2]NO3) solutions could prevent the aggregation of denatured hen egg-white lysozyme (HEWL). The researchers also demonstrated that it is possible to separate refolded active protein from [N0,0,0,2]NO3 with a simple desalination method. This approach allows the application of ILs in intermediate processing phases, even if the IL is inadequate for the final formulation. Since then, different research groups have studied ILs for protein stabilization, particularly considering the current efforts to replace organic solvents with greener alternatives for industrial processes (Kumar et al., 2017). However, not all ILs will be beneficial for proteins, and it is necessary to consider the characteristics and stability of the target protein, the properties of the ILs, and the application medium. Therefore, the following subsection will discuss the variables that can influence the interactions of ILs and proteins and how to assess these complex systems. 3.1. Interactions and effects Not all classes of ILs can guarantee protein stabilization or preserve their intrinsic biological activity. Some families of ILs interact negatively with the protein, impairing its stability or activity (Kumar et al., 2017), which is not necessarily bad since they can be used as inhibitors of undesired protein reactions. Additionally, even the same ILs can have distinct interactions with the protein according to the medium conditions and their concentration. For instance, the pH and temperature of the medium can alter the interactions between proteins and ILs and enhance or shift their effects (Noritomi et al., 2011; Veríssimo et al., 2021). Besides, depending on the ILs’ alkyl chain length, concentration, and medium temperature, some ILs can act either as a surface-active agent or an electrolyte. For short-chained ILs, an ionic salting-in/saltingout behavior will be prevalent (Miskolczy et al., 2004; Moniruzzaman et al., 2008). However, increasing the alkyl chain length of the IL cation or anion (C ≥ 7) will 13 Journal Pre-proof oo f enhance their surfactant properties and enable them to self-assemble above their critical micelle concentration (CMC) (Bowers et al., 2004; Buettner et al., 2022). This different concentration-dependence behavior will also impact the interactions between ILs and proteins, especially in more complex systems with other substances and variations of pH and temperature. Furthermore, these surface-active ionic liquids (SAILs) have an amphiphilic character, presenting both hydrophilic and hydrophobic functional groups (Veríssimo et al., 2022). Hence, as traditional surfactants, they can potentially be used as additives to improve the solubility of proteins with poor water solubility or to encapsulate biomolecules in their aggregates for drug delivery (Adawiyah et al., 2016; Buettner et al., 2022). Considering proteins also present both hydrophilic and lipophilic regions, SAILs can interact with different portions of the protein, to either disrupt their structure or stabilize it (Buettner et al., 2022). If the SAILs disrupt or stabilize the protein will depend on their structure and concentration (particularly if the concentration is below or above its CMC), and the protein’s structure and character (with high influence of its hydrophilicity or lipophilicity) (Buettner et al., 2022). Jo u rn al Pr e- pr The complexity of the protein-IL systems makes finding the best IL a challenge, in which to effectively select ILs to stabilize proteins, it is necessary to perform comprehensive studies that consider and balance the properties of ILs, intrinsic characteristics of the target protein, and medium conditions. Particularly for ILs, different properties and characteristics of their cation and anion pair (e.g., their nature and alkyl chain length) will determine their effect on protein structure and activity, including their hydrophobicity, polarity, pKa, concentration, stability, and hydrogenbonding capacity. (Patel et al., 2014) Depending on the properties of the protein and the IL, both cations and anions of the ILs can play a central role in stabilizing or destabilizing proteins (Baker et al., 2011). Regarding proteins, their size, hydrophobicity, isoelectric point (pI), charge, concentration, affinities, and stability will influence their interaction with the ions in solution (Manning et al., 2010, 1989). The surrounding medium has also a fundamental role in the synergy between ILs and proteins in solution, as well as pH, temperature, ionicity, and presence of other substances (Noritomi et al., 2011; Veríssimo et al., 2021). Note that some studies are using pure IL (solvent) for protein (solute) solubilization (Bihari et al., 2010; Li et al., 2019; Tamura et al., 2012), but most are still using ILs in aqueous or organic solutions. In addition to accounting for and controlling the different IL-protein systems variables, it is also useful to determine the types of IL-protein interactions that occur in each setting. A range of interactions can occur between ILs and proteins, namely electrostatic, hydrogen bonding, hydrophobic interactions, strong Coulomb interaction, and dispersion forces (Schröder, 2017). In particular, the charged, aromatic, or hydrophobic groups on the surface of dissolved proteins will propitiate the interactions between the IL ions in the solution and different protein regions. Thus, these groups will determine the potential of the IL to impair or improve the stability and function of the macromolecule (Reslan and Kayser, 2018; Sedlák et al., 2008). Furthermore, the proteins and ILs can interact with themselves (e.g., self-assembly) or with other substances in the solution (McManus et al., 2016; Miskolczy et al., 2004; Moniruzzaman et al., 2008). Hence, it is usually an intricate process to isolate and determine all the phenomena occurring between ILs and proteins in solution. Considering the complexity of IL-protein systems, a multi-technique approach is usually necessary to obtain an accurate panorama of their effects and interactions. For example, Bui-Le et al. (Bui-Le et al., 2020) studied the interaction of pyrrolidinium and 14 Journal Pre-proof imidazolium-based ILs with Superfolder Green Fluorescent Protein (sfGFP), demonstrating that it was necessary to apply multiple analytical methods to resolve their complex mechanisms at a molecular level. These authors were only able to understand the specific IL-sfGFP interactions after combining several structural characterization techniques, including ultraviolet-visible (UV-Vis), fluorescence, CD and NMR spectroscopies, and SAXS analysis (Bui-Le et al., 2020). This research depicts the need to consider many variables and technologies to determine the overall potential of different ILs classes as protein stabilizers. With this in mind and aiming at least to point out the main interactions, we will compile and discuss in the next subsection the effect of distinct IL families on the lipophilic and hydrophilic stability of non-enzymatic proteins. 3.2. Effect of ILs on non-enzymatic proteins Jo u rn al Pr e- pr oo f The current state-of-the-art is here organized according to the lipophilicity or hydrophilicity of the non-enzymatic proteins, being the studies presented in Tables 1 and 2, respectively. As aforementioned, herein we used the UniProt sequence of the most usual variant of each type of protein to calculate its grand average hydrophobicity index (GRAVY), molecular weight (MW), and instability index using the Expasy ProtParam tool (Artimo et al., 2012; ExPASy, 2018; UniProt, 2020). In GRAVY, values above 0 indicate the protein sequence is lipophilic, while values below zero are present in hydrophilic protein sequences. However, note that GRAVY represents the hydrophilicity of the protein sequence, not accounting for its secondary and tertiary structure, which are also crucial to determine if a protein will be water or liposoluble. Hemoglobin (Hb) for instance, even though it has a GRAVY slightly above 0 (0.017), this globular protein is still soluble in water (up to 20 mg.mL–1) (Sigma-Aldrich, 1996) as a result of its amphipathic character (i.e., it presents both hydrophobic and hydrophilic chains). In water, most of the sidechains exposed on the Hb surface are rich in nitrogen and oxygen atoms (polar groups), while the hydrophobic alkyl chains are buried inside the protein core (Lukin et al., 2003). That is why the protein is still soluble in water even with a GRAVY slightly above 0. Nevertheless, GRAVY is a useful tool to infer protein hydrophilicity and can help organize and analyze the vast data on ILprotein interactions. Finally, the instability index estimates the stability of proteins based on statistical analysis regarding the presence of certain dipeptides that are associated with unstable proteins (Guruprasad et al., 1990). Proteins with an instability index below 40 are considered stable. Tables 1 and 2 summarize the ILs effect on proteins according to the distinct IL classes, namely ammonium, cholinium, imidazolium, guanidinium, phosphonium, pyridinium and pyrrolidinium-based ILs, and provide the effect of the ILs on the stability of proteins [increase (↑), similar to control (=) or decrease (↓)] according to the concentration of ILs. 3.2.1. Effect of ILs on non-enzymatic lipophilic and amphipathic proteins Table 1 presents the properties and stability of lipophilic or amphipathic nonenzymatic proteins in presence of different IL solutions (i.e., IL classes and concentrations). 15 Journal Pre-proof Table 1. Stability (structural, thermal, aggregation, or simulation) of lipophilic or amphipathic proteins in different concentrations of ILs (neat or aqueous solutions). Specific information for each protein, namely, UniProt of the most usual variant, molecular weight (MW), instability index (II), and GRAVY‡ are also presented in the table. Protein ILs Insulin Ammoniumbased ILs UniProt: P01308 (A and B chains) [N0,2,2,2]PO₄, [N0,1,1,1]HSO4, [N0,2,2,2]SO₄, [N0,1,1,1]H2PO4, [N0,1,1,1][CH₃COO ] Concentration* Stability 0.5 - 2.0 M ↑ (Thermal, (Kumar and ↓aggregation) Venkatesu, 2013) oo f Cholinium-based ILs Ref. [Ch][Gln], [Ch]₂[Asn] 0.0008 M MW: 5.8 kDa [Ch][Asn] 0.0008 M II: 13.61 (stable) [Ch][Arg], [Ch]₂[Gln], [Ch][Lys] GRAVY: 0.218 [Ch][Gln], [Ch]₂[Asn], [Ch][Asn] e- pr (Human insulin) Pr 0.0008 M (Guncheva et al., 2019) = (Thermal) (Guncheva et al., 2019) ↓ (Thermal, (Guncheva et al., structural) 2019) ↓ (Structural) (Guncheva et al., 2019) al 0.0008 M ↑ (Thermal) rn Imidazoliumbased ILs [C₂MIm][CH₃CO O] 50 - 90 wt% (~ 3 ↑ (Simulation, (Li et al., 2019) - 6 M) thermal) (Li et al., 2019) [C₄MIm]Cl, [C₄MIm]Br (Kumar Venkatesu, 2014a) Jo u [C₂MIm][CH₃CO O], [C₄MIm]Cl, [C₄MIm]NO₃, [C₄MIm][CH₃SO₃] , [C₄MIm][N(CN)2] , [C₄MIm][CH₃CO ~100 % (~ 2 - 7 O], ↑ (Simulation) M) [C₆MIm][CH₃CO O], [C₈MIm][CH₃CO O], [C₁₀MIm][CH₃CO O], [C₁₂MIm][CH₃CO O] 0.01 - 0.04 M [C₄MIm][CH₃CO 0.3 M O], [C₄MIm][CF₃COO ↑ (Structural) and ↑ (Structural, (Todinova et al., thermal) 2016) 16 Journal Pre-proof ], [C₄MIm][N(CN)2] (ILs in KCl/HCl pH 2) [C₄MIm]Cl, [C₄MIm]Br ↓ (Thermal) (Kumar Venkatesu, 2014a) and 0.01 - 0.04 M [C₄MIm][SNC], [C₄MIm]HSO₄, [C₄MIm]I, [C₄MIm][CH₃CO O] (Kumar ↓ (Structural, Venkatesu, thermal) 2014a) and 0.01 - 0.04 M [C₄MIm][C(CN)₃] in KCl/HCl pH 2 0.3 M oo ↓ (Structural, 50 % (v/v) (~ thermal, (Jha et al., 2014) 4.5, 3, 2, 1.5 M) simulation) al Pr [N1,1,1,1]OH, [N2,2,2,2]OH, [N3,3,3,3]OH, UniProt: P69905 (A and C chains) [N4,4,4,4]OH and P68871(B and D chains) Imidazolium(Human Hb A) based ILs ↓ (Structural, (Todinova et al., thermal) 2016) pr Ammoniumbased ILs f = (Structural, (Todinova et al., thermal) 2016) e- Hemoglobin (Hb) [C₄MIm]Cl, [C₄MIm][SNC], 0.3 M (ILs in KCl/HCl pH 2) [AMIm]Cl GRAVY: 0.017 ‡ (Jha and Venkatesu, 2016) 0.00006 0.00223 M ↓ (Structural) (Vashishat et al., 2017) [C₁₂MIm]Cl 0.00059 - 0.2240 ↓ (Structural) M (Vashishat et al., 2017) [AMIm]Cl 0.15 - 0.25 M ↓ (Structural) (Jha and Venkatesu, 2016) rn II: 6.59 (Stable) - ↑ (Structural) [C₆MIm][C₁₀SO₄] Jo u MW: 64.5 kDa 0.01 - 0.10 M GRAVY - grand average hydrophobicity index, close to 0 indicates an amphipathic protein sequence and above a lipophilic protein sequence. *Approximate conversions (when possible) to molar (M) using MW and density (when available on the manufacturer’s site or the literature). 17 Journal Pre-proof As presented in Table 1, the most studied ILs for insulin and Hb stabilization were imidazolium-based ILs, followed by ammonium and cholinium ILs. The ILs’ influence on protein stabilization is dependent on their concentration, ranging from 10 -5 M (in water) to neat ILs. Certain ILs enhanced, maintained, or impaired the stability of the protein. However, their interactions and effect go further than increasing or decreasing the protein stability. Before discussing the specific protein-IL interactions, it is necessary to understand the protein's native structure, properties, and function. Therefore, Fig. 3.A presents the structure of insulin, and Fig. 3.B of Hb, with each protein discussed according to the properties detailed in Table 1. oo f Fig. 3. Structure of A) Insulin (Human insulin, PDB ID: 3E7Y) and B) Hemoglobin (Human hemoglobin A, PDB ID: 1MKO). Images of the proteins were produced with the PDB structures using UCSF Chimera 1.14 (Berman et al., 2002; Pettersen et al., 2004). 3.2.1.1. Insulin Jo u rn al Pr e- pr The hormone insulin was the first biopharmaceutical produced and applied in humans, being fundamental for the treatment of Diabetes mellitus (Mayer et al., 2007; Walsh, 2013). Despite being a small protein (around 12 kDa), insulin has the structural complexity of other larger proteins, with two polypeptide chains (A and B) linked by disulfide bonds (Brange and Langkjœr, 1993), as presented in Fig. 3.A (represented by the Human insulin, PDB ID: 3E7Y). The A chain comprises two antiparallel α-helices whereas the B chain possesses a single α-helix with a turn and β-strand (Brange and Langkjœr, 1993). Regarding insulin behavior in an aqueous solution, this protein is a weak dimer that presents both polar and non-polar residues, forming dimers above 10-6 M and larger aggregates as hexamers at 2 mM (Brange and Langkjœr, 1993). Furthermore, insulin presents low solubility in water at neutral pH (as expected from its positive 0.218 GRAVY), but it can be solubilized in acidic pH (2 to 3) up to 0.17 mM (Sigma-Aldrich, 2014). Considering the instability index, insulin displays a low index (II = 13.61), which means it is stable; so its products can be preserved without refrigeration for up to 28 days at 15 to 30 ºC (Center for Drug Evaluation and Research, 2018). However, if the insulin has been frozen, exposed to higher temperatures, or altered (e.g., diluted), it may lose its potency and should not be used (Center for Drug Evaluation and Research, 2018). Hence, in addition to insulin being an excellent model protein due to its small size and complete protein structure, there is still room to improve the stability of insulin products. Considering the pursuit of developing more stable insulin formulations, different groups have studied the effect of ILs on this protein. Kumar and Venkatesu (Kumar and Venkatesu, 2013) observed that highly concentrated (0.5 to 2.0 M) ammonium-based ILs aqueous solutions of triethylammonium phosphate ([N0,2,2,2]PO₄), trimethylammonium hydrogen sulfate ([N0,1,1,1]HSO4), triethylammonium sulfate ([N0,2,2,2]SO₄), trimethylammonium dihydrogen phosphate ([N0,1,1,1]H2PO4), and trimethylammonium acetate ([N0,1,1,1][CH₃COO]) prevented the self-aggregation of insulin into inactive forms while also increasing its thermal stability (Kumar and Venkatesu, 2013). In another approach, Guncheva et al. (Guncheva et al., 2019) concluded that highly dilute aqueous solutions (0.8 mM) of cholinium ILs had different impacts on insulin structure and its thermal stability [assessed by insulin Tm and ∆Hm], namely: aqueous solutions of cholinium L-arginate ([Ch][Arg]), dicholinium Lglutaminate ([Ch]₂[Gln]) and cholinium L-lysinate ([Ch][Lys]) led to a partial 18 Journal Pre-proof denaturation of insulin and decreased its thermal stability; cholinium L-asparaginate ([Ch][Asn]) was able to maintain insulin thermal stability despite causing a partial denaturation of the protein; cholinium L-glutaminate ([Ch][Gln]) and dicholinium Lasparaginate ([Ch]₂[Asn]) improved the thermal stability of insulin (Guncheva et al., 2019). al Pr e- pr oo f In addition to ammonium and cholinium ILs, there has been an even greater diversity of studies applying imidazolium-based ILs for insulin stabilization. Following this line of research, Li et al. (Li et al., 2019) used molecular dynamics (MD) to predict the stability of insulin in concentrated solutions of imidazolium ILs, namely: 1-ethyl-3methylimidazolium acetate ([C₂MIm][CH₃COO]), 1-butyl-3-methylimidazolium chloride ([C₄MIm]Cl), 1-butyl-3-methylimidazolium nitrate ([C₄MIm]NO₃), 1-butyl-3methylimidazolium methanesulfonate ([C₄MIm][CH₃SO₃]), 1-butyl-3methylimidazolium dicyanamide ([C₄MIm][N(CN)2]), 1-butyl-3-methylimidazolium acetate ([C₄MIm][CH₃COO]), 1-hexyl-3-methylimidazolium acetate ([C₆MIm][CH₃COO]), 1-octyl-3-methylimidazolium acetate ([C₈MIm][CH₃COO]), 1decyl-3-methylimidazolium acetate ([C₁₀MIm][CH₃COO]), and 1-dodecyl-3methylimidazolium acetate ([C₁₂MIm][CH₃COO]). The MD evaluation demonstrated that insulin is most stable in pure and 25 wt% hydrated imidazolium-based ILs with weak hydrogen bond basicity and shorter alkyl chains (Li et al., 2019). MD assays also showed that the protective effect of ILs on insulin derived from electrostatic interactions. These accounted for approximately 77 % of the interaction energy between ILs and insulin, against about 33 % of the van der Waals forces (Li et al., 2019). Finally, Li et al. (Li et al., 2019) performed experimental work using differential scanning micro-calorimetry (micro-DSC), confirming the thermal stability of insulin in [C₂MIm][CH₃COO] solutions of concentrations ranging from 50 to 90 wt% (Li et al., 2019). Jo u rn Similarly, Kumar and Venkatesu (Kumar and Venkatesu, 2014a) evaluated the effect of dilute aqueous solutions (0.01 to 0.04 M) of 1-butyl-3-methylimidazoliumbased ILs with different anions, including [C₄MIm]Cl, 1-butyl-3-methylimidazolium bromide ([C₄MIm]Br), 1-ethyl-3-methylimidazolium thiocyanate ([C₄MIm][SNC]), 1butyl-3-methylimidazolium hydrogensulfate ([C₄MIm]HSO₄), 1-butyl-3methylimidazolium iodine ([C₄MIm]I), and ([C₄MIm][CH₃COO]). From this experimental study, they observed that only [C₄MIm]Br and [C₄Mim]Cl stabilized the native state of insulin, while all the other anions denatured the protein. Interestingly, no link was observed between the Hofmeister series (which associates the potential of salt ions to solubilize and stabilize proteins) (Baldwin, 1996; Zhang and Cremer, 2006) and the effect of ILs on insulin structure, thus supporting the idea that IL-protein stabilization involves other variables and more complex mechanisms (Kumar and Venkatesu, 2014a). Furthermore, none of the ILs protected insulin against thermal denaturation (Kumar and Venkatesu, 2014a). Todinova et al. (Todinova et al., 2016) also studied dilute IL solutions but evaluated the potential of ILs to protect insulin structure under acidic conditions (pH 2). The ILs [C₄MIm][CH₃COO], 1-butyl-3methylimidazolium trifluoroacetate ([C₄MIm][CF₃COO]), [C₄MIm][N(CN)2], [C₄MIm]Cl, and [C₄MIm][SNC] preserved or enhanced insulin helical structure, with the first three ILs also improving protein thermal stability, while the last two ILs maintained the insulin Tm (Todinova et al., 2016). Nevertheless, the IL 1-ethyl-3methylimidazolium tricyanomethanide ([C₄MIm][C(CN)₃]) stimulated the formation of random coils and unordered forms, reducing insulin T m (Todinova et al., 2016). 19 Journal Pre-proof e- pr oo f In summary, all three classes of ILs discussed in the previous paragraphs presented compounds capable of improving or degrading insulin stability, depending on their anion and cation pair, concentration, and environmental conditions (e.g., temperature, pH). Overall, IL solutions with higher concentrations (above 0.5 M to neat ILs) improved insulin stability, while lower concentrations of ILs had mixed results depending on the constitution of the ILs. However, it would be necessary to evaluate the effect of low concentrations of ammonium ILs and high concentrations of cholinium ILs on insulin instability to confirm this trend, as there were no studies with these parameters found in the literature. Nevertheless, from this set of works, it is still unclear if there is a trend between the properties of the ions and their effect on protein stability, as they did not follow the Hofmeister series (Kumar and Venkatesu, 2014a). There are likely more complex mechanism and variables governing the IL-protein interactions that still requires further evaluation to set a trend regarding the IL composition and its effect on insulin. Furthermore, these works also show that certain IL (e.g., [Ch][Asn]) can improve insulin thermal stability despite causing partial denaturation of the protein (Guncheva et al., 2019), while certain imidazolium ILs (e.g., [C₄MIm]Cl, and [C₄MIm][SNC]) stabilized the native state of insulin but had no impact on insulin T m (Kumar and Venkatesu, 2014a). Hence, it is necessary to understand that the same IL can be considered a “stabilizer” by a specific parameter (e.g., maintenance of the native structure, Tm, activity, aggregation rate) but a “destabilizer” if the metric evaluated changes. 3.2.1.2. Hemoglobin (Hb) Jo u rn al Pr Hemoglobin (Hb) is a metalloprotein of red blood cells that transports oxygen in almost all vertebrates (Giardina et al., 1995). Currently, there are also attempts to develop Hb-based blood substitutes for transfusions, though it is still necessary to overcome the toxicity and accumulation of cell-free Hb in the blood (Center for Biologics Evaluation and Research, 2020). Furthermore, Hb and other heme proteins can be used to develop catalysis biomaterials (Huang et al., 2011; Wang et al., 2007, 2005). As seen in Fig. 3.B, the Hb structure has four subunits (α1, α2, β1, and β2 chains), each containing one polypeptide chain and one heme group, which is a ringlike organic structure (porphyrin) with an iron atom (Marengo-Rowe, 2006). The arrangement of Hb quaternary structure (subunits) differs in the presence or absence of oxygen through a reversible bond to the iron atom, i.e., oxyhemoglobin (red) and deoxyhemoglobin (purple-blue), respectively (Marengo-Rowe, 2006). As stated before, Hb is amphipathic, with mainly hydrophilic amino acids on its surface, while most of the hydrophobic chains are buried inside the protein when exposed to aqueous environments (Lukin et al., 2003). Many non-covalent interactions are required to maintain the complex Hb tetrameric structure, including hydrophilic and hydrophobic forces, hydrogen bonds, van der Waals forces, and electrostatic interactions (Vashishat et al., 2017; Wang et al., 1999). Therefore, Hb is very susceptible to denaturation or conformational changes that can impair its medical and biotechnological applications. Different studies evaluated the effect of ILs on Hb structure and function to improve its stability or develop Hb materials and formulations. For instance, Jha and Venkatesu (Jha et al., 2014) studied the stabilizing aptitude of ammonium-based ILs, showing that concentrated ILs solutions [50 % (v/v)] decreased the thermal stability and changed the secondary structure of Hb and another heme protein, Myoglobin (Mb). The ILs included tetramethylammonium hydroxide ([N1,1,1,1]OH), tetraethylammonium hydroxide ([N2,2,2,2]OH), tetrapropylammonium hydroxide ([N3,3,3,3]OH) and tetrabutylammonium hydroxide ([N4,4,4,4]OH). Among these ILs, the ones with shorter 20 Journal Pre-proof e- pr oo f alkyl chains such as [N1,1,1,1]OH were stronger destabilizers of the heme proteins than the bulkier ILs like [N4,4,4,4]OH, following the trend [N1,1,1,1]OH > [N2,2,2,2]OH > [N3,3,3,3]OH > [N4,4,4,4]OH. Interestingly, this study confirmed that the cation alkyl chain length also plays a role in IL-protein interactions in addition to the usual dominant anion effect on protein stability (Jha et al., 2014). In that case, the authors suggested that the unfolding of Hb and Mb could be a result of favorable interactions between the functional groups of the protein's surface and the IL ions. Using Molecular Docking (PatchDocking), the researchers concluded that the increase of the destabilizing effect of cations with shorter alkyl chains could be due to easier access of smaller cations to interact with the hydrophobic residues of Hb and Mb. For example, the more polarized tetramethyl group allowed a stronger bond between the protein surface and the IL cation, while the ILs with longer cationic alkyl side chains had reduced contact with the Hb surface (Jha et al., 2014). In another work, these authors (Jha and Venkatesu, 2016) assessed the IL impact on Hb structure and stability, namely the effect of different 1allyl-3-methylimidazolium chloride ([AMIm]Cl) concentrations on Hb stabilization. While lower concentrations (0.01 to 0.10 M) stabilized Hb native structure, higher concentrations (0.15 to 0.25 M) had a detrimental effect (Jha and Venkatesu, 2016). The stabilization effect was explained as an accumulation of the [AMIm]+ cation on the protein surface while Cl- remained in the bulk phase, affecting the hydrogen bonding of Hb with the surrounding water molecules and thus stabilizing the Hb (Jha and Venkatesu, 2016). rn al Pr A study by Vashishat et al. (Vashishat et al., 2017), using surface-active imidazolium ILs, showed that even dilute solutions of 1-hexyl-3-methylimidazolium dodecyl sulfate ([C₆MIm][C₁₀SO₄]) and 1-dodecyl-3-methylimidazolium chloride ([C₁₂MIm]Cl) at concentrations as low as 0.06 to 2.23 mM and 0.59 to 224 mM, respectively, impaired Hb stability. These results suggest that lower concentrations of [C₆MIm][C₁₀SO₄] form stronger IL-Hb monomer complexes than [C₁₂MIm]Cl. However, at higher concentrations, [C₁₂MIm]Cl causes more Hb denaturation, even inducing the release of the heme group from the hydrophobic pocket of Hb. Jo u Overall, all ammonium and imidazolium-based ILs reduced Hb stability (Jha et al., 2014; Vashishat et al., 2017), except for very dilute solutions of [AMIm]Cl (0.01 to 0.10 M) (Jha and Venkatesu, 2016). However, even a slight increase in the concentration of [AMIm]Cl (from 0.15 to 0.25 M) impaired Hb stability (Jha and Venkatesu, 2016). Considering Hb's complex quaternary structure, with four chains and an MW of 64.5 kDa, compared to the simpler structure of insulin with two chains and an MW of 5.8 kDa, Hb was more susceptible to structural changes and decreased of its thermal stability by ILs than insulin. Among the ammonium ILs, the ones with shorter alkyl chains were stronger destabilizers than the bulkier counterparts, following the trend [N1,1,1,1]OH > [N2,2,2,2]OH > [N3,3,3,3]OH > [N4,4,4,4]OH (Jha et al., 2014). This effect is likely due to easier access to smaller cations to interact with the hydrophobic residues of Hb (Jha et al., 2014). Furthermore, very dilute surface-active imidazolium ILs solutions ([C₆MIm][C₁₀SO₄] and[C₁₂MIm]Cl) reduced Hb stability (Vashishat et al., 2017). As discussed before, Hb has an overall hydrophobic primary chain, being soluble in water because its hydrophobic chains are buried inside the protein in aqueous environments. Hence, the presence of hydrophobic compounds can expose Hb hydrophobic residues and disrupt its quaternary structure. In this subsection, we discussed the current state-of-the-art of ILs application for the stabilization of lipophilic (insulin) and amphipathic (Hb) non-enzymatic proteins. In the next subsection, we will dive even further to understand the IL-protein interactions 21 Journal Pre-proof of hydrophilic proteins, as the research using water-soluble proteins and ILs is more extensive and diverse. 3.2.2. Effect of ILs on non-enzymatic hydrophilic proteins Jo u rn al Pr e- pr oo f Table 2 presents the properties and stability of non-enzymatic hydrophilic proteins in different IL classes and concentrations. 22 Journal Pre-proof Table 2. Stability (structural, thermal, activity, aggregation, long-term, chemical stress, or simulation) of lipophilic or amphipathic proteins in different concentrations of ILs. Specific information for each protein, namely, UniProt of the most usual variant, molecular weight (MW), instability index (II), and GRAVY‡ are also presented in the table. Protein ILs Concentration* Stability Ref. 0.001 M ↑ (Structural) (Jaganathan et al., 2015) [N0,0,0,2]NO3 0.1 - 2.5 M = (Structural) (Baker et al., 2011) [N0,0,0,2][CHO2], [N0,0,0,1][CHO2] 50 - 70 % = (Structural) (Wei and Danielson, 2011) [N0,0,0,2][CHO2], [N0,0,0,1][CHO2] 20 % [N0,0,0,2][CHO2], [N0,0,0,1][CHO2] 80 % MW: 12.4 kDa e- II: 15.51 (stable) Cholinium-based ILs GRAVY: -0.875 [Ch]H2PO4 = (Structural, (Wei and Danielson, thermal) 2011) oo (Cyt C from horse heart) ↓ (Structural, (Wei and Danielson, activity) 2011) pr UniProt: P00004 [N0,0,0,2]NO3 f Cytochrome C Ammonium-based ILs (Cyt C) 70 wt% (~ 3.5 M) ↑ (Structural) (Fujita and Ohno, 2010) ↑ (Structural, (Fujita et al., 2007, thermal, activity, 2006, 2005) long-term) 20 wt% (~ 1 M) ↓ (Thermal) 80 wt% (~ 3 M) ↓ (Structural, (Fujita et al., 2007) activity) [AMIm]Cl ~100 % ↑ (Structural, (Tamura thermal, 2012) activity) [C₂MIm][Tf₂N] ~100 % ↑ (Structural) (Ciaccafava et al., 2011) [C₂MIm][EtSO₄] ~100 % ↑ (Activity) (Bihari et al., 2010) [C₄MIm]Cl 25 % (w/v) (1.4 = (Structural) M) Pr 80 wt% (~ 4 M) [Ch]H2PO4 al [Ch]H2PO4 rn [Ch][(CH3(CH2)3)2HPO4] (Fujita et al., 2005) Jo u Imidazolium-based ILs et al., (Baker and Heller, 2009) [C₄MIm]Cl, [C₄MIm]Br, [C₄MIm][N(CN)2], [C₄MIm]BF₄, > 0.25 M [C₄MIm]NO₃, [C₂MIm][CH₃COO] = (Structural) (Baker et al., 2011) [C₄MIm]Cl 0.24 - 0.45 M = (Structural) (Baker et al., 2011) [C₂MIm][Tf₂N] ~100 % ↓ (Activity) (Ciaccafava et al., 2011) ↓ (Structural) (Baker et al., 2011) [C₄MIm]Cl, [C₄MIm][N(CN)2], [C₄MIm]Br, ~ 0.5 - 2.5 M [C₄MIm]BF₄, 23 Journal Pre-proof [C₄MIm]NO₃, [C₂MIm][CH₃COO] [C₂MIm][CH₃SO₃] ~ 0.50 - 1.25 M ↓ (Structural) (Baker et al., 2011) [C₂MIm][EtSO₄] ~100 % ↓ (Structural) (Bihari et al., 2010) [C₅MIm]Br 0.9 - 1.5 M ↓ (Structural) (Sen Mojumdar et al., 2012) [C₄MIm]Cl 50 % (w/v) (~ 3 ↓ (Structural) M) (Baker and Heller, 2009) [C₄MIm]Cl 1 - 30 mol% (~ 0.5 ↓ (Structural) - 5 M) (Takekiyo 2014b) [C₄MIm][CH₃COO], [C₄MIm][Lac], [C₄MIm][MeSO₄] 80 wt% al., (Fujita et al., 2007) oo f ↓ (Structural) et pr Pyridinium- and pyrrolidiniumbased ILs ↑ (Thermal) (Fujita et al., 2006, 2005) = (Structural) (Fujita et al., 2006, 2005) 80 wt% (~ 4 M) ↓ (Activity) (Fujita et al., 2007) 20 wt% (~ 1 M) ↓ (Thermal) (Fujita et al., 2005) 1 mol% (~ 0.5 M) ↑ (Activity, (Han et al., 2021) ↓aggregation) UniProt wtGFP: [N0,2,2,2][CH₃SO₃] P42212 1 mol% (~ 0.5 M) ↑ (Activity, (Han et al., 2021) ↓aggregation) MW: 26.9 kDa [N0,0,0,4]NO3 1 - 5 mol% (~ 0.5 ↓ (Activity, (Han et al., 2021) - 2 M) ↑aggregation) II 31.07 (stable) [N0,0,0,2][CH₃SO₃] 5 -17 mol% (~ 2 - ↓ (Activity, (Han et al., 2021) 4.5 M) ↑aggregation) GRAVY -0.521 [N0,0,0,2]NO3 1 - 17 mol% (~ 0.5 ↓ (Activity, (Han et al., 2021) - 6 M) ↑aggregation) [N0,0,0,(2OH)]NO3 1 - 5 mol% (~ 0.5 ↓ (Activity, (Han et al., 2021) - 2.3 M) ↑aggregation) [N0,2,2,2][CH₃SO₃] 5 -17 mol% (~ 2 - ↓ (Activity, (Han et al., 2021) 3.7 M) ↑aggregation) 80 wt% (~ 4 M) e- [C4C1Pyr]H2PO4 80 wt% (~ 4 M) Pr [C4C1Pyr]H2PO4 [C4C1Pyr]H2PO4 al [C4C1Pyr]H2PO4 Ammonium-based ILs Variant sfGFP [N0,0,0,2][CH₃SO₃] Variant sfGFP Jo u rn Green Fluorescent Protein (GFP) Cholinium-based ILs [Ch][CH₃COO] 1 mol% (~ 0.5 M) ↑ (Activity, (Han et al., 2021) ↓aggregation) 24 Journal Pre-proof [Ch]H2PO4 1 - 17 mol% (~ 0.5 ↑ (Activity, (Han et al., 2021) - 4.7 M) ↓aggregation) [Ch][CH₃SO₃] 1 - 5 mol% (~ 0.5 ↑ (Activity, (Han et al., 2021) - 2 M) ↓aggregation) [Ch][CH₃COO] 5 - 10 mol% (~ 3.5 ↓ (Activity, (Han et al., 2021) - 5 M) ↑aggregation) Imidazolium-based ILs [C₁C1Im]Cl, [C₂MIm]Cl, [C₄MIm]Cl, [C₆MIm]Cl, 0.025 - 0.500 M [C₈MIm]Cl, [C₁₀MIm]Cl ↑ (Activity, (Veríssimo et al., chemical stress, 2021) long-term) Variant wtGFP [C₄MIm]Cl 1.56 M ↑ (Structural, ↓aggregation, (Heller et al., 2010) activity) Variant EGFP [C₁₂MIm]Cl 0.025 - 0.100 M Variant wtGFP [C₄MIm]Cl 3.12 M Variant wtGFP [C₄MIm]Cl Variant EGFP [C₁₂MIm]Cl Variant sfGFP [C₄MIm]Cl, [C₄MIm][CH₃COO], 1M [C₄MIm][TfO] oo f Variant EGFP pr ↑ (Activity, (Veríssimo et al., chemical stress, 2021) long-term) e- ↓ (Structural, (Heller et al., 2010) activity) ↓ (Thermal) 1.56 - 3.12 M (Heller et al., 2010) Pr ↓ (Activity, (Veríssimo et al., chemical stress, 2021) long-term) 0.25 - 0.50 M al ↓ (Structural, thermal, (Bui-Le et al., 2020) activity) rn Pyridinium- and pyrrolidiniumbased ILs [C4C1Pyrr]Cl, [C4C1Pyrr][CH₃COO], [C4C1Pyrr][TfO] Myoglobin (Mb) Ammonium-based ILs Jo u Variant sfGFP ↓ (Structural, thermal, (Bui-Le et al., 2020) activity) 1M [N0,0,2,2]SO₄, [N0,0,2,2]PO₄, UniProt: P02144 [N0,2,2,2]SO₄, [N0,2,2,2]PO₄, 50 % (v/v) [N0,1,1,1]HSO4, [N0,1,1,1]H2PO4 ↑ (Structural, (Attri et al., 2014) thermal) (Human Mb) [N1,1,1,1]OH, [N2,2,2,2]OH, 50 % (v/v) [N3,3,3,3]OH, [N4,4,4,4]OH ↓ (Structural, thermal, (Jha et al., 2014) simulation) MW: 17.2 kDa [N0,2,2,2][CH₃COO], [N0,0,2,2][CH₃COO], [N0,1,1,1][CH₃COO] ↓ (Structural, (Attri et al., 2014) thermal) 50 % (v/v) II: 21.81 (stable) Imidazolium-based ILs GRAVY: -0.476 [C₂MIm][Phe] 0.000005 0.00005 M - = (Structural) (Sankaranarayanan et al., 2012) 25 Journal Pre-proof 0.22 M = (Structural) [C₂MIm][CH₃COO] 0.05 - 0.15 M = (Structural, (Fiebig et al., 2014) chemical stress) [C₄MIm]BF₄ 0.05 - 0.15 M ↓ (Structural, thermal, (Fiebig et al., 2014) chemical stress) [C₂MIm][Phe] 0.00005 - 0.0010 ↓ (Structural) M [C₄MIm][SNC], [C₄MIm]HSO₄, [C₄MIm]Cl, [C₄MIm]Br, 0.01 - 0.04 M [C₄MIm][CH₃COO], [C₄MIm]I ↓ (Structural, (Kumar and thermal, Venkatesu, 2014b) simulation) oo 0.025 - 1.00 % (w/v) (0.001 – ↑ (Structural, (Rawat and Bohidar, 0.04 M) ↓aggregation) 2015, 2012) pr UniProt: P02769 [C₈MIm]Cl [C₄MIm]Cl = (Structural) (Du et al., 2007) 0.0005 – 0.0030 M = (Structural) (Lin et al., 2013) al [EtOCOCH₂MIm][C₁₀SO₄] Pr e- ~ 0.6 M II: 40.11 [C₈MIm]Br (unstable) GRAVY: -0.475 [C₄MIm]BF₄, [C₄MIm]PF₆ [C₄MIm]NO₃, rn [C₄MIm]Cl, [C4C4Im]Cl (Sankaranarayanan et al., 2012) f Bovine Serum Imidazolium-based ILs Albumin (BSA) MW: 66.4 kDa (Safavi and Farjami, 2010) [C₄MIm]Cl 0.00002 – 0.001 M = (Structural, (Wang et al., 2012) thermal) < 0.030 M ↓ (Structural, (Zhu et al., 2011) thermal) 0.0005 – 0.0020 M ↓ (Structural, (Shu et al., 2011) simulation) Jo u [C₄MIm]Br, [C₆MIm]Br, ↓ (Structural, 0.0005 – 0.0080 M (Yan et al., 2012) [C₈MIm]Br, [C₁₀MIm]Cl simulation) [C₄MIm]Cl, [C₈MIm]Cl [C₆MIm]Cl, 0.000025 0.000150 M - ↓ (Structural) (Huang et al., 2013) [C₄MIm][C₈SO₄], [C₈MIm]Cl 0.00031 - 0.00044 ↓ (Structural) M [C₁₄MIm]Br 0.00003 – 0.10000 ↓ (Structural, (Geng et al., 2010, M thermal) 2009) [EtOCOCH₂MIm][C₁₀SO₄] 0.001 – 0.00500 M ↓ (Structural, (Wang et al., 2012) thermal) 20 wt% (~ 1 M) = (Structural) (Chen et al., 2014) 0.0015 – 0.0040 M = (Structural) (Ding et al., 2014) (Singh et al., 2012) Ammonium-based ILs [N0,1,1,(2OH)][C2CO2] Guanidinium-based ILs [diHOHTMGu]Cl, [TMGu][CH₃CH₂COOH] 26 Journal Pre-proof Phosphonium-based ILs 20 % (w/v) (~ 0.5 ↓ (Structural, (Li and Wu, 2014) M) thermal) [P4,4,4,4][SS] Pyridinium- and pyrrolidiniumbased ILs [EtOCOCH₂Pyrr][C₁₀SO₄] 0.00002 – 0.001 M = (Structural) [EtOCOCH₂Pyrr][C₁₀SO₄] 0.001 – 0.00500 M [C4C1OPyrr]Br 0.00415 0.02920 M ↓ (Structural, (Wang et al., 2012) thermal) – ↓ (Structural, (Kumari simulation) 2014a) et al., et al., oo f Human Serum Cholinium-based ILs Albumin (HSA) 20 % (v/v) (~ 1.5 ↑ (Structural) M) UniProt: P02768 [Ch]H2PO4 Imidazolium-based ILs (Akdogan 2011) pr MW: 66.5 kDa (Wang et al., 2012) 0.2 % (w/v) (0.008 ↑ (Structural, (Rawat and Bohidar, M|) ↓aggregation) 2012) e- II: 38.85 (stable) [C₈MIm]Cl 25 % (w/v) (1.4 = (Structural) M) Pr GRAVY: -0.395 [C₄MIm]Cl al [C₂MIm][N(CN)2], [C₂MIm][Me₂PO₄], [C₂MIm]SCN, 35 % (v/v) [C₂MIm]BF₄, [C₂MIm]NO₃, [C₂MIm][EtSO₄] ↓ (Structural) (Baker and Heller, 2009) (Akdogan and Hinderberger, 2011) 15 - 50 % (v/v) (~ ↓ (Structural) 1 - 3 M) [C₅MIm]Br 0.9 M ↓ (Structural) (Kumar Das et al., 2012) [C₅MIm]Cl 0.3 - 1.5 M ↓ (Structural) (Sasmal et al., 2011) [C₄MIm]Cl 50 % (w/v) (3 M) ↓ (Structural) (Baker and Heller, 2009) 70 - 98 % (v/v) ↓ (Structural) (Page et al., 2009) Jo u rn [C₂MIm]BF₄, [C₆MIm]BF₄ [C₄MIm][Tf₂N], [C₄MIm]PF₆ [C₄MIm]BF₄, [OHC2MIm]Cl, [C₂OCMIm]Cl, [C₂MIm]Cl, [C₄MIm]Cl, 0.005 M [C₄MIm][N(CN)2], [C4C4Im]Cl (Akdogan 2011) et al., ↓ (Structural, (Silva et al., 2014) thermal) Pyridinium- and pyrrolidiniumbased ILs [C4C1OPyrr]Br 0.0167 – 0.1044 M ↓ (Structural, (Kumari simulation) 2014b) et al., β-Lactoglobulin Ammonium-based ILs (BLG) UniProt: P02754 [N0,2,2,2][CH₃SO₃] 20 - 80 wt% (~ 1 - ↑ (Thermal) (Byrne et al., 2013) 27 Journal Pre-proof 4 M) (Bovine BLG) [N0,2,2,2][CH₃SO₃] 20 wt% (~ 1 M) ↑ (Structural) (Byrne et al., 2013) MW: 18.4 kDa [N0,2,2,2]PO₄, [N0,2,2,2]SO₄ 20 - 80 wt% (~ 1 = (Structural) 4 M) (Byrne et al., 2013) II: 42.43 [N0,2,2,2][CH₃SO₃] (Unstable) 40 - 80 wt% (~2 – ↓ (Structural) 4 M) (Byrne et al., 2013) GRAVY: -0.162 [N0,0,0,2]NO3 15 - 50 mol% (~ ↓ (Structural) 5.5 - 9.5 M) (Takekiyo 2013) 20 - 80 wt% (~ 1 ↓ (Structural) 4 M) (Byrne et al., 2013) [N0,2,2,2][CF₃COO] et al., Imidazolium-based ILs 15 - 20 mol% (~ ↓ (Structural, (Takekiyo et 3.5 - 4.5 M) ↑aggregation) 2014a, 2013) [C₂MIm][EtSO₄] 0.001 - 0.010 M oo f [C₂MIm]NO₃, [C₄MIm]NO₃ (Sankaranarayanan et al., 2013) pr ↓ (Structural) al., ‡ Jo u rn al Pr e- GRAVY - grand average hydrophobicity index, below 0 indicates that the protein sequence is hydrophilic. *Approximate conversions (when possible) to molar (M) using MW and density (when available on the manufacturer’s site or literature). 28 Journal Pre-proof Table 2 clearly shows that there is considerably more research done with nonenzymatic hydrophilic proteins than for their lipophilic counterparts. Besides, there is also a higher diversity in the IL classes used for stabilizing this class of proteins. As with lipophilic and amphipathic proteins, imidazolium-based ILs are the most studied, followed by ammonium and cholinium IL families. However, there are also studies using guanidium, phosphonium, pyridinium and pyrrolidinium-based ILs. Once more, all scenarios were observed with these proteins that had their stability enhanced or decreased depending on the IL and its concentration range (from around 10-6 M to neat ILs). Each hydrophilic protein under study has been displayed in Fig. 4, its function, properties, and native structure are discussed in the next subsections as well as the ILprotein synergies. pr oo f Fig. 4. Structure of the hydrophilic proteins A) Cytochrome C from horse heart (PDB ID: 1HRC), B) Wild-type Green Fluorescent Protein (PDB ID: 1GFL), C) Human myoglobin mutant K45R (PDB ID: 3RGK), D) Bovine serum albumin (PDB ID: 4F5S), E) Human serum albumin (PDB ID: 1BM0), and F) Bovine β-Lactoglobulin A (PDB ID: 1CJ5). Images of the proteins were produced with the PDB structures using UCSF Chimera 1.14 (Berman et al., 2002; Pettersen et al., 2004). 3.2.2.1. Cytochrome C (Cyt C) Jo u rn al Pr e- Cytochrome C (Cyt C) is a small and highly soluble heme protein (around 12 kDa) necessary for adenosine triphosphate (ATP) synthesis in mitochondria, while also being a component of the respiratory electron transport chain between complexes III and IV (Bertini et al., 2006). This protein is also associated with programmed cell death since, after an apoptotic stimulus, Cyt C is released into the cytosol triggering apoptosis (Ow et al., 2008). Moreover, Cyt C can catalyze redox reactions, such as hydroxylation and aromatic oxidation, which brings an industrial interest in using it to develop catalytic materials (Wang et al., 2005). As seen in Fig. 4.A, Cyt C has an α-helical core with a heme prosthetic group attached by two thioether covalent bonds to cysteine residues in the protein. Cyt C is not only a stable protein but is also capable of refolding after denaturation under many conditions. Therefore, it is also widely used as a model for protein folding studies. Considering the influence of ammonium-based ILs on Cyt C stability, both Baker et al. (Baker et al., 2011) and Jaganathan et al. (Jaganathan et al., 2015) reported the ability of ILs to maintain Cyt C structure in presence of 0.1-2.5 M and 0.001 M of [N0,0,0,2]NO3 aqueous solutions, respectively. Furthermore, Jaganathan et al. (Jaganathan et al., 2015) found that dilute [N0,0,0,2]NO3 solutions make Cyt C native structure more compact and resistant to denaturation, helping the protein folding and refolding. The authors suggested that [N0,0,0,2]NO3 assists in Cyt C renaturation and protection against urea denaturation by forming a tightly organized assembly around the protein structure (Jaganathan et al., 2015). Likewise, Wei and Danielson (Wei and Danielson, 2011) observed that ethylammonium formate ([N0,0,0,2][CHO2]) and methylammonium formate ([N0,0,0,1][CHO2]) maintained Cyt C conformation at room temperature when present in concentrations between 50 to 70 wt% (Wei and Danielson, 2011). Additionally, 20 wt% of [N0,0,0,2][CHO2] and [N0,0,0,1][CHO2] preserved Cyt C structure from 30 to 50 ºC, while 80 wt% of each IL changed Cyt C secondary structure and decreased its activity to one-third of its initial (Wei and Danielson, 2011). Different studies have demonstrated the neutral or positive effects of high concentrations (70 - 80 wt%) of cholinium dihydrogen phosphate ([Ch]H2PO4) on Cyt 29 Journal Pre-proof pr oo f C stability (Fujita et al., 2007, 2006, 2005; Fujita and Ohno, 2010). In contrast, low concentrations (20 wt%) of [Ch]H2PO4 (Fujita and Ohno, 2010) or high concentrations (80 wt%) of cholinium dibutylphosphate ([Ch][(CH3(CH2)3)2HPO4]) (Fujita et al., 2007) impaired Cyt C stability. In 2005, Fujita et al. (Fujita et al., 2005) observed that 80 wt% of [Ch]H2PO4 solubilizes around 3 mM of Cyt C, obtaining a protective effect against thermal denaturation up to 100 ºC, which is higher than the 75 ºC observed in the presence of sodium phosphate buffer. However, there is a decrease in Cyt C thermal stability when exposed to a 20 wt% aqueous solution of [Ch]H2PO4 (Fujita et al., 2005). In the following study, Fujita et al. (Fujita et al., 2006) demonstrated that 80 wt% of [Ch]H2PO4 in an aqueous solution can maintain the native structure and activity of Cyt C after six months of room temperature storage, against only one week in sodium phosphate or Tris-acetate buffers. The authors related this positive effect to a decline in protein hydrolysis in the concentrated IL (Fujita et al., 2006). Later, the same authors (Fujita et al., 2007) demonstrated that [Ch]H2PO4 maintained Cyt C activity and native structure for 18 months at room temperature since this IL presented an excellent combination between a chaotropic cation and a kosmotropic anion, which guaranteed a good protein solubility and stability. Lastly, Fujita and Ohno (Fujita and Ohno, 2010) showed that a 70 wt% of [Ch]H2PO4 in water can be applied to solubilize and maintain/preserve other metalloproteins beyond Cyt C, including peroxidase, ascorbate oxidase, azurin, pseudoazurin, and fructose dehydrogenase. Jo u rn al Pr e- In addition to the studies using cholinium-based ILs, Fujita et al. (Fujita et al., 2007, 2006, 2005) also evaluated the effect of high and low concentrations of pyridinium-based ILs on Cyt C. As reported by them, high concentrations (80 wt%) of 1-butyl-1-methylpyridinium dihydrogen phosphate ([C4C1Pyr]H2PO4) preserved Cyt C native structure and increased its thermal stability (Fujita et al., 2007, 2006, 2005). However, 80 wt% of [C4C1Pyr]H2PO4 decreased the superoxide reduction activity of Cyt C (Fujita et al., 2007). Furthermore, 20 wt% of [C4C1Pyr]H2PO4 impaired the thermal stability of Cyt C, following the behavior previously observed with 20 wt% of [Ch]H2PO4 (Fujita et al., 2005). The similarities of the impact of [Ch]H2PO4 and [C4C1Pyr]H2PO4 at both 20 and 80 wt% indicates a dominant anion influence of the H2PO4− on Cyt C structural stability. When imidazolium-based ILs were considered as stabilizing agents for Cyt C, the previous studies were once again very diverse. Tamura et al. (Tamura et al., 2012) observed that neat [AMIm]Cl is a suitable solvent for Cyt C at 80 ºC, also maintaining 90 % and 75 % of its redox activity after 3 h at 120 and 140 ºC, respectively. In comparison, Cyt C completely lost its activity after 1 h at 50 ºC in a buffer solution (Tamura et al., 2012), which confirms the strong stabilizing aptitude of [AMIm]Cl. In another study with neat ILs, Ciaccafava et al. (Ciaccafava et al., 2011) showed that the hydrophobic non-water-miscible IL 1-ethyl-3-methylimidazolium bis(trifluoromethylsulfonyl)imide ([C₂MIm][Tf₂N]) stabilized Cyt C native structure. Moreover, [C₂MIm][Tf₂N] increased 30-fold the electroactivity of Cyt C but at the cost of its catalytic oxidation of H2 via hydrogenases. The authors suggested that the neat ILs can inhibit the hydrogenases, affecting Cyt C catalytic activity (Ciaccafava et al., 2011). Nevertheless, if 20 wt% of buffer solution was added to the ILs, the inhibition of the hydrogenases was prevented, allowing the development of IL-Cyt C electrolytes for biofuel cell design (Ciaccafava et al., 2011). On the other hand, Bihari et al. (Bihari et al., 2010) and Fujita et al. (Fujita et al., 2007) observed that a high concentration of IL solutions impaired the structural stability of Cyt C. Bihari et al. (Bihari et al., 2010) demonstrated that the neat IL 130 Journal Pre-proof oo f ethyl-3-methyl imidazolium ethylsulfate ([C₂MIm][EtSO₄]) caused alterations in Cyt C structure similar to acid denaturation in a buffer. Nonetheless, this also led to a 3-fold increase in the peroxidase activity of Cyt C. This increase can be explained due to a Cyt C protein structure alteration, particularly the perturbation or loss of Met80 as an axial ligand to the heme group. The authors explained that the heme group in native Cyt C is buried inside the protein crevice, and all of its six ferric coordinate bonds are occupied. Hence, the loss of its tertiary structure exposes the heme group, like in other genuine heme peroxidase enzymes (e.g., horse-radish peroxidase), increasing its peroxidase activity (Bihari et al., 2010). As for Fujita et al. (Fujita et al., 2007), they showed that aqueous solutions of 80 wt% of [C₄MIm][CH₃COO], 1-butyl-3-methylimidazolium lactate ([C₄MIm][Lac]), and 1-butyl-3-methylimidazolium methylsulfate ([C₄MIm][MeSO₄]) reduced Cyt C superoxide reduction activity. Overall, [C₄MIm][MeSO₄], followed by [C₄MIm][Lac] and [C₄MIm][CH₃COO], caused the greatest decrease in Cyt C activity, while, as shown above, [Ch]H2PO4 had no negative impact on the protein. Authors suggested that the anion’s kosmotropicity impacted the decrease of Cyt C activity, since the least kosmotropic ILs caused the most intense inhibition. Jo u rn al Pr e- pr As for more diluted IL solutions, other authors also concluded that different concentrations of imidazolium ILs have distinct impacts on Cyt C. Baker and Heller (Baker and Heller, 2009) observed that a 10 % (w/v) [C₄MIm]Cl solution (0.6 M) preserved Cyt C secondary structure, while 50 % (w/v) of [C₄MIm]Cl (3 M) denatured the protein. Baker et al. (Baker et al., 2011) demonstrated that the use of a [C₄MIm]Cl aqueous solution lower than 0.5 M and [C₄MIm]Cl, [C₄MIm]Br, [C₄MIm][N(CN)2], 1butyl-2,3-dimethylimidazolium tetrafluoroborate ([C₄MIm]BF₄), [C₄MIm]NO₃, [C₂MIm][CH₃COO] lower than 0.25 M preserved Cyt C structure. Nonetheless, [C₄MIm]Cl, [C₄MIm]Br, [C₄MIm][N(CN)2], [C₄MIm]BF₄, [C₄MIm]NO₃, [C₂MIm][CH₃COO] from 0.50 to 2.50 M and 1-ethyl-3-methylimidazolium methanesulfonate ([C₂MIm][CH₃SO₃]) from 0.50 to 1.25 M altered Cyt C structure. Specifically, Takekiyo et al. (Takekiyo et al., 2014b) observed that [C₄MIm]Cl solutions from 1 to 30 mol% (0.15 to 4.5 M) disrupted the tertiary structure of Cyt C. However, above 10 mol% (> 1.5 M), there was an interesting refolding of the protein αhelical structure, i.e., the protein started to regain its native α-helical form (Takekiyo et al., 2014b). Sen Mojumdar et al. (Sen Mojumdar et al., 2012) also noticed that adding 0.9 and 1.5 M of 1-pentyl-3-methylimidazolium bromide ([C₅MIm]Br) to Cyt C caused an increase in its hydrodynamic radius, suggesting a partial unfolding of the protein. Overall, highly dilute ammonium ILs solutions increased Cyt C stability, intermediate solutions maintained Cyt C activity whereas high concentrations decreased the protein stability (Baker et al., 2011; Jaganathan et al., 2015; Wei and Danielson, 2011). For cholinium-based ILs, high concentrations of [Ch][(CH3(CH2)3)2HPO4] also led to a detrimental effect on Cyt C structure (Fujita et al., 2007). Yet, for both [Ch]H2PO4 and the pyridinium IL [C4C1Pyr]H2PO4, high concentrations (80 wt%) of IL improved Cyt C stability while lower (20 wt%) concentrations impaired it, showing a dominant anion effect (Fujita et al., 2007, 2006, 2005; Fujita and Ohno, 2010). In contrast, for imidazolium-based ILs, there was a vast range of outcomes: i) very dilute solutions had no impact on Cyt C stability (Baker et al., 2011), ii) intermediate concentrations either maintained (Baker and Heller, 2009) or impaired (Baker and Heller, 2009; Baker et al., 2011; Sen Mojumdar et al., 2012; Takekiyo et al., 2014a) the stability of the protein, and iii) high concentration of ILs were reported to increase 31 Journal Pre-proof (Bihari et al., 2010; Ciaccafava et al., 2011; Tamura et al., 2012) or decrease the protein stability (Bihari et al., 2010; Fujita et al., 2007). 3.2.2.2. Green fluorescent proteins (GFP) Pr e- pr oo f Green fluorescent proteins (GFP) present intense and natural fluorescence, allowing their application as biomarkers and biosensors (Tsien, 1998; Zimmer, 2002). There is a wide range of GFP variants with slight modifications to their structure and distinct fluorescence attributes, physical-chemical properties, and stabilities (Zimmer, 2002). GFP variants present the same overall structure, with one main β-barrel with a core helix maintaining the chromophore, as presented in Fig. 4.B. These variants are highly soluble and most are weak dimers, being found as monomers or dimers in solution depending on their concentration and environmental conditions (Lambert, 2019). The chromophore of GFP mutants is easily accessible to several external disturbances, such as pH, temperature, and certain substances (Ward et al., 1982). Once the GFP is denatured, there is a disruption of the cylindrical protein structure that holds the chromophore at its center and, consequently, occurs a fluorescence extinction (Ward et al., 1982). Therefore, GFP and its variants emit fluorescence only when their protein structure is intact (Enoki et al., 2004). Each GFP variant has a different resistance to physical and chemical stresses (Tsien, 1998; Zimmer, 2002). For instance, most GFP mutants are relatively resistant to photobleaching and thermal, chemical, and biological denaturation (Cubitt et al., 1995; Tsien, 1998; Zimmer, 2002), but have distinct sensitivities to pH and oxidizing agents (Mazzola et al., 2006; Santos et al., 2007; Zimmer, 2002). Hence, solvents can be added to different GFP formulations to improve their stability or act as biosensors’ modulators by quenching and dequenching their fluorescence. Jo u rn al As aforementioned, many GFP variants are weak dimers prone to aggregation, which usually leads to fluorescence quenching. Aiming to find solvents that protect GFP from loss of activity, Han et al. (Han et al., 2021) monitored the effect of a series of ILs aqueous solutions on sfGFP fluorescence and aggregation behavior. They observed a decrease of sfGFP aggregation in the presence of IL solutions, namely ethylammonium methanesulfonate ([N0,0,0,2][CH₃SO₃]), triethylammonium methanesulfonate ([N0,2,2,2][CH₃SO₃]) and cholinium acetate ([Ch][CH3COO]) at 1 mol%, [Ch]H2PO4 from 1 to 17 mol% and cholinium methanesulfonate ([Ch][CH₃SO₃]) from 1 to 5 mol%. Contrarily, butylammonium nitrate ([N0,0,0,4]NO3) and ethanolammonium nitrate ([N0,0,0,(2OH)]NO3) at 1-5 mol%, [N0,0,0,2][CH₃SO₃], [N0,2,2,2][CH₃SO₃] and [Ch][CH3COO] from 5 to 17 mol% and [N0,0,0,2]NO3 from 1 to 17 mol% increased protein aggregation. Overall, i) the nitrate anion quenched sfGFP fluorescence and led to a less compact structure; ii) the methanesulfonate anion was able to maintain sfGFP fluorescence and its globular structure when prepared in solutions of ethylammonium and cholinium ILs as well as [N0,2,2,2][CH₃SO₃] at low concentrations; and iii) high concentrations of [N0,2,2,2][CH₃SO₃] completely suppressed the fluorescence of the fluorophore. This phenomenon confirms that the cation also plays a role in the influence of ILs on proteins. Moreover, Han et al. (Han et al., 2021) indicated that these ILs can establish weak hydrogen bonds with sfGFP at its surface, which simultaneously hides its hydrophobic groups away from the water molecules, hence enhancing sfGFP hydration and stabilizing the water-protein interface. Other fundamental aspects of protein application are the long-term preservation at room temperature and the resistance of their products to chemical stresses. With this in mind, Veríssimo et al. (Veríssimo et al., 2021) evaluated the aptitude of imidazolium-based ILs to preserve the Enhanced GFP (EGFP) fluorescence activity at 32 Journal Pre-proof pr oo f short-term (48 h), long-term (3 months), and under chemical stress (e.g., acid, surfactant, and oxidizing agents). The ILs 1,3-dimethylimidazolium chloride ([C₁C1Im]Cl), 1-ethyl-3-methylimidazolium chloride ([C₂MIm]Cl), [C₄MIm]Cl, 1hexyl-3-methylimidazolium chloride ([C₆MIm]Cl), 1-octyl-3-methylimidazolium chloride ([C₈MIm]Cl), 1-decyl-3-methylimidazolium chloride ([C₁₀MIm]Cl) from 0.025 to 0.500 M, and [C₁₂MIm]Cl from 0.025 to 0.100 M maintained or improved the EGFP fluorescence activity under 48 h. However, [C₁₂MIm]Cl from 0.250 to 0.500 M impaired EGFP fluorescence in the short-term study. At 0.100 M, all the ILs preserved EGFP fluorescence for the three months study versus one week with the control solution, i.e., NaCl 0.100 M in water. ILs can help stabilize EGFP by reducing its aggregation, though it is still necessary to confirm this mechanism. Moreover, all IL solutions at 0.1 M were able to protect EGFP from sodium dodecyl sulfate (SDS) denaturation in comparison with the control solutions, namely sodium phosphate buffer (PBS) and sodium chloride (NaCl). Besides, the authors showed that ILs with shorter cation alkyl chains ([CnMIm]Cl, n = 2, 4, 6, and 8) can also protect EGFP against hydrogen peroxide (H₂O₂) and guanidinium hydrochloride (GuHCl) (Veríssimo et al., 2021). Together these works confirm that diluted solutions of imidazolium ILs have great potential to act as stabilizers of EGFP formulations. al Pr e- In another study with imidazolium ILs and GFP, Heller et al. (Heller et al., 2010) showed that a 1.56 M aqueous solution of [C₄MIm]Cl increases wild-type GFP (wtGFP) stability while decreasing it at 3.12 M. In this work, authors demonstrated that [C₄MIm]Cl favors the monomeric state of wtGFP and reduces aggregation. However, it also makes the protein structure less compact and more prone to thermal denaturation. This effect was sharper at 3.12 M (highest concentration), showing a concentrationdependent nature. Therefore, at lower concentrations (1.56 M), [C₄MIm]Cl improved the stability by reducing wtGFP aggregation; however, at higher concentrations (3.12 M), the detrimental denaturation effect of [C₄MIm]Cl on wtGFP structure was dominant. Jo u rn Considering the complex nature of IL-protein interactions, Bui-Le et al. (Bui-Le et al., 2020) used a multi-technique approach and sfGFP as a model protein to better understand the effects and synergy between ILs and proteins. The results showed a decrease in sfGFP thermal stability for 1 M solutions of the imidazolium ILs [C₄MIm]Cl, [C₄MIm][CH₃COO], and 1-butyl-3-methylimidazolium trifluoromethanesulfonate ([C₄MIm][TfO]), and the pyrrolidinium-based ILs 1-butyl-4methyl pyrrolidinium chloride ([C4C1Pyrr]Cl), 1-butyl-4-methyl pyrrolidinium acetate ([C4C1Pyrr][CH₃COO]) and 1-butyl-1-methylpyrrolidinium trifluoromethanesulfonate ([C4C1Pyrr][TfO]). The anions had a dominant effect on the destabilization of sfGFP, with similar results obtained for both [C₄MIm] + and [C4C1Pyrr]+ cations. Both Cl− and [CH₃COO]− decreased the Tm of sfGFP by reducing the enthalpic barrier to the denaturation of the protein tertiary structure, hence weakening the interactions that maintain its tertiary structure. In contrast, [TfO]− increased the entropic gain from denaturation by contracting the protein structure and reducing the barrier to unfolding as a result of the preferential interactions with the protein surface, particularly with hydrophobic residues (Bui-Le et al., 2020). As observed from the previous studies with ILs and GFP, there is a fine balance between the concentration of the ILs and the improvement of the protein stability by reducing its aggregation, or GFP destabilization by making its structure less compact (Han et al., 2021; Heller et al., 2010; Veríssimo et al., 2021). The nature of the anion (Bui-Le et al., 2020; Han et al., 2021) and the length of the cation alkyl side chain (Han 33 Journal Pre-proof et al., 2021; Veríssimo et al., 2021) played a role in enhancing or reducing GFP stability. In general, lower concentrations of IL solutions and shorter cation alkyl chains had a more positive impact on GFP stability than higher IL concentrations and longer alkyl chains. It is also interesting to note that considering GFP application as a biosensor, even ILs that quench GFP fluorescence can be used as additives to modulate its use in biosensing, particularly if they cause reversible changes to GFP structure. Hence, future studies that consider the reversibility of the effects of certain ILs on GFP fluorescence activity and structure could help to expand the applications of this protein. 3.2.2.3. Myoglobin (Mb) al Pr e- pr oo f Myoglobin (Mb) is another heme protein found primarily in the striated muscle of vertebrates, functioning as an oxygen-storage unit for myocytes (Wittenberg and Wittenberg, 2003). Mb can retain oxygen as its heme group can bind and release O2 depending on the concentration of gas in the cell (Wittenberg and Wittenberg, 2003). It is also associated with the hemostasis of nitric oxide and the detoxification of reactive oxygen species (Wittenberg and Wittenberg, 2003). Mb is one of the most studied proteins, being the first to have its three-dimensional structure revealed by X-ray crystallography (Ordway and Garry, 2004), particularly, as a model for globular proteins. As presented in Fig. 4.C, Mb comprises a globin (a single polypeptide chain with eight α-helices) and one heme group (Hubbard et al., 1990). It has a higher affinity for oxygen than Hb, and it is also smaller and more soluble in water (Sigma-Aldrich, 2018). Although Mb is considerably stable, substitutions in its H64L and H64F residues lead to mutants 10 to 30 types more stable, suggesting that the stability of the protein is sacrificed to maintain the distal histidine (H64) while increasing oxygen affinity and inhibiting auto-oxidation (Hargrove et al., 1994). Hence, it is possible to stabilize Mb, although proper assessment of whether modifications in its environment impair its function is crucial, particularly in IL-protein studies. Jo u rn Aiming to understand the effect of high concentrations [50 % (v/v)] of ammonium-based ILs on Mb stability and structure, Attri et al. (Attri et al., 2014) selected ILs with different anions (sulfate, phosphate, and acetate) and ammonium cations (diethylammonium and triethylammonium) while Jha et al. (Jha et al., 2014) maintained the anion (hydroxide) and altered the ammonium cation. Attri et al. observed that using solutions at 50 % (v/v) of ILs with sulfate and phosphate anions [[N0,2,2,2]PO₄, diethylammonium phosphate ([N0,0,2,2]PO4), [N0,1,1,1]H2PO4, [N0,2,2,2]SO4, diethylammonium sulfate ([N0,0,2,2]SO4) and [N0,1,1,1]HSO4 increased the thermal stability of Mb, following the order: [N0,2,2,2]PO4 > [N0,0,2,2]PO4 > [N0,1,1,1]H2PO4 > [N0,2,2,2]SO4 > [N0,0,2,2]SO4 > [N0,1,1,1]HSO4 (Attri et al., 2014). On the other hand, IL solutions with acetate anions (trimethylammonium acetate ([N0,1,1,1][CH3COO]), triethylammonium acetate ([N0,2,2,2][CH₃COO]) and diethylammonium acetate ([N0,0,2,2][CH3COO]) at 50 % (v/v) decreased the protein Tm, with [N0,1,1,1][CH3COO] being the most destabilizer, followed by [N0,2,2,2][CH3COO] and then [N0,0,2,2][CH3COO]. As stated in other studies, it is an overall assumption that the anion presents a dominant effect on the stabilization or destabilization of the protein, yet the cation still plays an important role. Interestingly, in the work of Attri et al. (Attri et al., 2014), the intensity order of the anion effect did not follow the Hofmeister series trend. Regarding the Mb structure, the ILs acting as thermal stabilizers increased or maintained the α-helix (%) of the protein in comparison with the buffer, while the destabilizers reduced it (Attri et al., 2014). Considering that the native state of Mb comprises eight α-helices and a heme group, the sulfate and phosphate ILs preserved the protein structure even better than the buffer, while the acetate-based ILs caused the 34 Journal Pre-proof oo f unfolding of Mb. Authors suggested that the sulfate and phosphate anions (stabilizers) have unfavorable interactions with the Mb surface, being repelled and forming a hydration layer around the protein. This phenomenon forces the polypeptide to adopt a more compact folded structure, increasing Mb stability. As for the ILs with an acetate anion, i.e. [N0,1,1,1][CH3COO], [N0,2,2,2][CH₃COO] and [N0,0,2,2][CH3COO], these have stronger interactions with the Mb surface, perturbing its internal protein bonds and causing the protein to unfold (Attri et al., 2014). As for Jha et al. (Jha et al., 2014), 50 % (v/v) solutions of [N1,1,1,1]OH, [N2,2,2,2]OH, [N3,3,3,3]OH, and [N4,4,4,4]OH reduced Hb and Mb thermal and structural stability, respectively. Using molecular docking tools, the authors showed that shorter alkyl chain cations had easier access to the protein’s hydrophobic groups, enhancing Hb and Mb unfolding. Interestingly, despite the differences in Hb and Mb size and hydrophobicity (with Hb being four times larger and more amphipathic), the ILs had a similar effect on both globular proteins. Therefore, comparing these two studies, using high concentrations of ammonium ILs [50 % (v/v)], the changes in the cations and anions of ammonium-based ILs will alter its effect on Mb, considering Attri et al. (Attri et al., 2014) saw an increased in the thermal and structural stability of Mb while Jha et al. observed the opposite effect (Jha et al., 2014) rn al Pr e- pr As for imidazolium-ILs, Sankaranarayanan et al. (Sankaranarayanan et al., 2012) evaluated the effect of highly dilute (0.005 to 1 mM) solutions of imidazoliumand amino acid-based ILs, i.e., 1-ethyl-3-methylimidazolium phenylalanine ([C₂MIm][Phe]), on Mb structural stability. From 0.005 to 0.05 mM, [C₂MIm][Phe] preserved Mb native structure and between 0.05 to 0.2 mM, [C₂MIm][Phe] completely altered the native helical form of Mb to β-sheet. Nevertheless, diluting this sample allowed the protein to change the β-sheet and return to its original α-helix form. When Mb was left for one week on the IL at 0.2 mM, its β-sheet conformation started to organize itself as micrometer-sized fibrils (self-assembly), a phenomenon not observed for Mb in water after one week. Above 0.2 mM, the protein changed from β-sheet to random coiled structures, showing even further loss of Mb native structure. The Mb fibrils formed with the addition of ILs have potential applications for the development of novel bio-based ILs materials. Jo u Also employing imidazolium-based ILs, Fiebig et al. (Fiebig et al., 2014) showed that low concentrations (0.05 to 0.15 M) of [C2 MIm][CH3COO] maintain Mb structural stability, while [C₄MIm]BF₄ impairs it. Despite not unfolding the protein, [C₄MIm]BF₄ reduced the threshold for Mb denaturation by the acid GuHCl when compared to Mb and GuHCl in only phosphate buffer. For example, [C₄MIm]BF₄ decreased the required concentration and variations in ΔG for Mb unfolding, i.e., 80 % decrease, from 44 to 8 kJ.mol–1. Furthermore, they also evaluated the effect of the salts sodium acetate and lithium tetrafluoroborate to verify the contribution of the anion on Mb stability, identifying similar results to the ILs. Hence, it was suggested a dominant effect of the anion's capacity to interact with the Mb surface. These researchers speculated that BF₄–, as a polarizable anion, can interact with the surface of the protein and infiltrate its inner hydrophobic core, disrupting the hydrogen bonds and other weaker interactions while maintaining the Mb structure stable. In another approach using imidazolium-based ILs, Kumar and Venkatesu (Kumar and Venkatesu, 2014b) tried to assess if there was a relationship between the effect of IL anion in Mb stability and the Hofmeister series. They evaluated dilute IL solutions (0.01 to 0.04 M) of [C₄MIm][SNC], [C₄MIm]HSO₄, [C₄MIm]Cl, [C₄MIm]Br, [C₄MIm][CH₃COO] and [C₄MIm]I, as well as a set of their corresponding ionic salts (sodium salts with different anions) as control samples. Both ILs and salts decreased 35 Journal Pre-proof f Mb thermal stability, while higher concentrations of the ionic species increased the flexibility of the Mb structure. Again, there was no relationship between the Hofmeister series and the impact of the ions on Mb stability. However, molecular docking results suggested that the [C₄MIm]+ cation disturbs the interactions between the His97 and the heme group, forming a cavity and facilitating the anion's access to disturb the environment around the porphyrin. Interestingly, the cation had no direct interactions with the amino acid residues. In presence of the heme group, [CH₃COO]− interacts with amino acid residues of the cavity formed by [C₄MIm] +. On the other hand, the SO₄−2 anion does not directly interact with amino acid residues, likely due to hydrophobic interactions, but it is close to the heme group. As for the [SCN]−, as it is highly charged and small, this anion suffers less from steric repulsion and can interact directly with the heme group and other amino acid residues in the region. The destabilization of Mb in these ILs is a result of the individual contributions of cations and anions in solutions, particularly due to distinct interactions between the ions and the protein surface. Pr e- pr oo Overall, more IL solutions impaired Mb stability than maintained or enhanced it (Table 2), similar to what occurred to the other heme-protein, Hb. Furthermore, the anion choice was decisive for the positive or negative effect of ILs on Mb structure, with ILs with the same cation and similar concentrations improving or decreasing Mb stability depending on the anion (Attri et al., 2014; Fiebig et al., 2014; Jha et al., 2014; Kumar and Venkatesu, 2014b; Safavi and Farjami, 2010). However, it is important to note that, although to a less extent, the cation can also interact with the protein or modulate the anion effect (Attri et al., 2014; Jha et al., 2014; Kumar and Venkatesu, 2014b). The concentration range is also relevant to define if the same IL preserved or reduced Mb stability (Kumar and Venkatesu, 2014b; Safavi and Farjami, 2010; Sankaranarayanan et al., 2012). al 3.2.2.4. Serum albumin Jo u rn Serum albumin (SA) is the most abundant plasma protein in the blood of mammals, being the primary carrier of fatty acids in the bloodstream (Majorek et al., 2012). This protein can also bind non-specifically to other molecules, such as a diversity of steroids, metabolites, and pharmaceuticals (Stillwell, 2016). SA is a globular, hydrophilic, and relatively small (MW of around 65 kDa, depending on the species) unglycosylated protein (Belinskaia et al., 2021). As presented in Fig. 4.D and 4.E, the SA structure has several long α-helices in a unique sequence of disulfide double loops, which makes its structure rigid (Hubbard et al., 1990; Majorek et al., 2012). SA has three homologous domains numbered I, II, and III, subdivided into A and B subdomains. SA also presents 11 hydrophobic binding domains that allow this protein to carry multiple fatty acids (Belinskaia et al., 2021). Moreover, the SA structure can vary slightly for each mammal (Peters Jr, 1995). The two most studied, and with more commercial applications, are the bovine serum albumin (BSA) and the human serum albumin (HSA) (Jahanban-Esfahlan et al., 2019). 3.2.2.4.1. Bovine serum albumin (BSA) BSA has a vast number of uses, including i) standards for protein quantification; ii) template to synthesize nanostructures; iii) drug carrier; ingredient/additive of culture medium; and iv) reactant in several biochemical assays (e.g., immunoblots, immunohistochemistry, and enzyme-linked immunosorbent assay) (Ma et al., 2020; Solanki et al., 2021; Wang and Zhang, 2018). Regarding its stability, lyophilized BSA is stable for three weeks at room temperature, but its recommended use after reconstitution is 2 to 7 days if stored at 4 ºC (Prospec, 2022). Due to its low cost, 36 Journal Pre-proof accessibility and wide range of applications, BSA is a suitable model protein for studies of protein-ILs interactions. pr oo f As presented in Table 2, most studies using ILs and BSA resulted in either preservation or impairment of BSA stability. However, two previous studies reported a specific stabilization of BSA and HSA dispersions close to their pI due to aggregation inhibition. Particularly, Rawat and Bohidar (Rawat and Bohidar, 2012) observed that low concentrations of [C₈MIm]Cl [0.025 to 0.250 % (w/v), i.e., 0.001 to 0.01 M] preserved the secondary structure of BSA and HSA and reduced protein aggregation even in a pH close to their pI. Additionally, their results suggested that a selective binding of the ILs to the surface of the protein creates an IL-bilayer on the protein monomers, contributing to the stability of BSA and HSA dispersions. Due to its charge and ability to establish H-bonds, [C₈MIm]Cl may penetrate the first hydration layer of BSA and form the IL bilayer, stabilizing both protein monomers. In a followed-up study (Rawat and Bohidar, 2015), these researchers observed a similar result for higher concentrations of [CnMIm]Cl aqueous solutions [namely, n = 2, 4, 6, and 8, at 1 % (w/v), i.e., 0.04 M] not only for the inhibition of BSA aggregation but also of βLactoglobulin (BLG) and immunoglobulin (IgG) proteins. The best stabilizer and aggregation inhibitor for BSA was [C₈MIm]Cl, while [C₂MIm]Cl provided better results for BLG and IgG (Rawat and Bohidar, 2015). al Pr e- Different studies using IL-based aqueous biphasic systems (ABS) for BSA extraction have also included some information regarding the preservation of protein structure at low concentrations of imidazolium-based ILs. Du et al. (Du et al., 2007) applied an ABS with approximately 0.6 M of [C₄MIm]Cl with dipotassium hydrogen phosphate (K₂HPO₄) and Lin et al. (Lin et al., 2013) used 0.5 to 3 mM of [C₈MIm]Br and potassium dihydrogen phosphate (KH₂PO₄) for BSA extraction, both confirming the integrity of the protein after its extraction. Jo u rn Researchers also evaluated how changing the anion of imidazolium-based ILs affected the structural stability of BSA. Zhu et al. (Zhu et al., 2011) reported that diluted aqueous solutions (<0.030 M) of [C₄MIm]BF₄ and 1-butyl-3-methylimidazolium hexafluorophosphate ([C₄MIm]PF₆) changed the secondary structure of BSA, with similar results for both ILs. A thermodynamic analysis of the IL-BSA interactions revealed two types of interactions with specific binding sites on the protein for the cation. The high-affinity binding was due to electrostatic interaction with negatively charged sites on the BSA surface, while the low affinity occurred between the imidazole ring and the hydrophobic cavity of the protein (Zhu et al., 2011). The thermodynamic results also indicated a high-affinity interaction (electrostatic) of the anions with positively charged residues on the BSA surface (Zhu et al., 2011). Similarly, Shu et al. (Shu et al., 2011) demonstrated that dilute IL solutions (0.5 to 2 mM) of [C₄MIm]Cl, [C₄MIm]NO₃ and 1,3-dibutylimidazolium chloride ([C4C4Im]Cl) led to BSA unfolding, mainly due to electrostatic and hydrophobic interactions between the ILs and the protein polypeptides. These researchers observed that [C₄MIm]NO₃ altered the most BSA structure, with [C₄MIm]Cl and [C4C4Im]Cl presenting similar results, as revealed by molecular docking that showed that the IL cations specifically interact with the hydrophobic residues of BSA domain III. Considering the influence of the cation on BSA structure, other research groups aimed to elucidate the effect of increasing the length in the cation alkyl side chain of the ILs on BSA stability. As with the previous studies, Huang and co-workers (Huang et al., 2013) reported that the imidazolium ILs, namely [C₄MIm]Cl, [C₆MIm]Cl, and [C₈MIm]Cl, at 0.025 - 0.150 mM, changed the secondary structure of BSA as a result of 37 Journal Pre-proof interactions with the internal hydrophobic residues of the protein. Furthermore, they found that by increasing the cation alkyl side chain length, the binding between the ILs and BSA is strengthened. Likewise, Yan et al. (Yan et al., 2012) observed that the ILs [C₄MIm]Br, 1-hexyl-3-methylimidazolium bromide ([C₆MIm]Br), 1-octyl-3methylimidazolium bromide ([C₈MIm]Br) and [C₁₀MIm]Cl, at 0.5 - 8 mM, changed the BSA structure. Hence, it was demonstrated that the cationic head groups of these imidazolium-based ILs can interact with the asparagine and glutamic acid residues on the BSA surface, while their alkyl side chains interact with the hydrophobic residues on the BSA core. The thermodynamic parameters suggested that the hydrophobic interactions played a major role in the interactions between [C₁₀MIm]Br and BSA. As for the [Cn mim]Br with n = 4, 6, and 8, the hydrogen bond and van der Waals forces were crucial for the IL-BSA binding. Once more, the increase in the cationic alkyl chain length resulted in larger alterations in BSA secondary structure. al Pr e- pr oo f Other reports are confirming the destabilizing effect of ILs with longer cationic alkyl side chains (surface-active ILs) on BSA. For example, Geng et al. (Geng et al., 2010, 2009) demonstrated that even low concentrations (0.03 to 10 mM) of 1tetradecyl-3-methylimidazolium bromide ([C₁₄MIm]Br) can induce BSA unfolding. As discussed in the previous studies, the cation interactions with internal hydrophobic residues of the protein are the main cause of BSA unfolding with the IL (Geng et al., 2010, 2009). Both the concentrations of [C₁₄MIm]Br and BSA influence the IL-protein interaction, and higher BSA concentrations increase the CMC of the IL as a result of the IL binding to BSA before micellization (e.g., IL CMC of 0.0028 M for 2 × 10−6 M of BSA, and IL CMC 0.0033 M for 5 × 10−5 M of BSA) (Geng et al., 2009). At concentrations lower than its CMC (< 0.003 M), [C₁₄MIm]Br interacts through electrostatic attraction and caused slight protein unfolding. Above its CMC (> 0.003 M), [C₁₄MIm]Br bounded to BSA by hydrophobic interactions and causes protein denaturation. Jo u rn In addition to studies regarding the effect of modification in the IL cation, there are also reports studying the impact of surfactant anions on BSA stability. Wang et al. (Wang et al., 2012) showed the denaturation of BSA with the ester-functionalized anionic SAILs, namely using aqueous solutions (from 1 to 5 mM) of 3-methyl-1(ethoxycarbonylmethyl)imidazolium dodecyl sulfate ([EtOCOCH₂MIm][C₁₀SO₄]) and 3-methyl-1-(ethoxycarbonylmethyl)pyrrolidinium dodecyl sulfate ([EtOCOCH₂Pyrr][C₁₀SO₄]). The ILs did not change de secondary structure of BSA below 1 mM but altered it with the increase of concentration to 1 to 5 Mm. However, when comparing imidazolium and pyrrolidinium SAILs, it was clear that the imidazolium SAIL [EtOCOCH₂MIm][C₁₀SO₄] caused a more profound alteration to the secondary structure of BSA than the pyrrolidinium SAIL (Wang et al., 2012). Like the previous studies using imidazolium-based ILs and BSA, the protein unfolding occurred by exposure of its internal hydrophobic residues. In another approach, Singh et al. (Singh et al., 2012) investigated the effect of [C₈MIm]Cl and 1-butyl-3methylimidazolium octyl sulfate ([C₄MIm][C₈SO₄]) on BSA structure from 0.31 to 0.44 mM. At low concentrations, [C₈MIm]Cl and [C₄MIm][C₈SO₄] have electrostatic interactions with BSA and cause its unfolding, with stronger binding for [C₈MIm]Cl. At higher concentrations (close to 0.44 mM), the hydrophobic interactions are dominant. There is also a dominance of hydrophobic interactions between BSA and [C₄MIm][C₈SO₄], while electrostatic forces are more present between [C₈MIm]Cl and the protein. 38 Journal Pre-proof On the other hand, two studies reported the preservation of the protein structure after BSA extraction using guanidinium-ILs-based ABS (Chen et al., 2014; Ding et al., 2014). Chen and co-workers (Chen et al., 2014) used an ABS composed of 20 wt% of 1,1-dimethylethanolaminium propanoate ([N0,1,1,(2OH)][C2CO2]) and K₂HPO₄ for the partition of BSA, confirming the integrity of the BSA structure after its protein partitioning. Ding et al. (Ding et al., 2014) also confirmed that BSA maintained its native structure after applying an ABS composed of 3.5 mM of N,N,N,N-tetramethylN,N-hexanol-guanidinium chloride ([diHOHTMGu]Cl) or tetramethylguanidinium methylacetate ([TMGu][CH₃CH₂COOH]) and K₂HPO₄ (at 3 M). e- pr oo f The effect of other IL families, such as phosphonium- and pyrrolidinium-based ILs, on BSA stability is also described in the literature. Li and Wu (Li and Wu, 2014) observed that the thermo-responsive IL tetrabutylphosphonium styrenesulfonate ([P4,4,4,4][SS]) at 20 % (w/v) reduced the thermal stability of BSA. These researchers suggested that both IL-protein interactions and the phase transition behavior of [P4,4,4,4][SS] are responsible for boosting the thermal denaturation of BSA. In another study, Kumari et al. (Kumari et al., 2014a) revealed BSA unfolding in presence of dilute 1-butyl-1-methyl-2-oxopyrrolidinium bromide [C4C1OPyrr]Br) aqueous solutions (4.15 to 29.2 mM), demonstrating a central role of hydrophobic forces in the IL-BSA binding. Specifically, the molecular modeling study revealed that [C4C1OPyrr]Br binds to BSA at the interface of subdomains IIA and IIIA mainly by hydrophobic interaction followed by hydrogen bonding. Jo u rn al Pr In summary, most of the works revealed that hydrophobic forces are usually responsible for BSA-ILs interactions, particularly for ILs with longer cation alkyl side chains and at higher IL concentrations (Geng et al., 2010, 2009; Kumari et al., 2014a; Singh et al., 2012). The overall destabilizing effect of ILs on BSA is usually due to the interaction of the ions with the BSA internal hydrophobic pocket, exposing hydrophobic residues that are buried in the BSA native state and causing the unfolding of this globular protein (Geng et al., 2010, 2009; Kumari et al., 2014a; Shu et al., 2011; Singh et al., 2012). Most studies cite the interaction of IL cations with the domains II and III of BSA (Kumari et al., 2014a; Shu et al., 2011). Electrostatic interactions, hydrogen bonding, and Van der Wall forces are also relevant in the effect of ILs on BSA for more diluted solutions and ILs with shorter alkyl chains (Geng et al., 2010, 2009; Kumari et al., 2014a; Shu et al., 2011; Singh et al., 2012). 3.2.2.4.2. Human serum albumin (HSA) HSA is the most abundant protein in human blood (Fanali et al., 2012). In addition to presenting similar properties and potential applications to BSA, HSA is less allergenic to humans and preferred for pharmaceutical formulations (Chruszcz et al., 2013). However, HSA has a higher cost than BSA (Bahreinipour et al., 2021), limiting its use outside the medical field. Due to its ability to bind to various molecules, including pharmaceuticals, HSA can be used as a drug carrier in medicines (Kouchakzadeh et al., 2014). Furthermore, the pharmaceutical industry also applies HSA as a vaccine stabilizer (Prymula et al., 2016). Like BSA, HSA follows the overall structure of serum albumins, as previously explained. Though, there are subtle differences in hydrophobicity between these proteins that can affect their properties and interactions with other substances (Maier et al., 2021). Considering the HSA relevance in the pharmaceutical industry, the study of HSA-IL interactions can facilitate the development of novel formulations for protein stabilization or drug delivery. For example, an earlier study by Akdogan et al. (Akdogan et al., 2011) reinforced the high biocompatibility of cholinium-based ILs with HSA, by preserving its native structure. A 39 Journal Pre-proof solution of HSA in 20% (v/v) of [Ch]H2PO4 more closely resembles the crystalline structure of the protein than the formulation in water, suggesting that [Ch]H2PO4 stabilizes its tertiary structure. Pr e- pr oo f For imidazolium-based ILs, the concentration and nature of each IL will define whether they preserve or denature HSA. For instance, Baker and Heller (Baker and Heller, 2009) demonstrated that in solutions up to 25 % (v/v) of [C₄MIm]Cl, HSA maintained its native tertiary structure; however, at 50 % (v/v), [C₄MIm]Cl led to HSA denaturation in a similar way to other protein denaturing agents, such as GuHCl and urea, leading to the formation of random coils. Furthermore, HSA dimerizes in [C₄MIm]Cl, something that does not occur with the protein in GuHCl. Additionally, [C₄MIm]Cl at 50 % (v/v) maintains Cyt C in its monomeric form, showing that dimerization is a process specific for HSA at high concentrations of [C₄MIm]Cl. Rawat and Bohidar (Rawat and Bohidar, 2012) observed that [C₈MIm]Cl from 1 to 10 mM preserved the secondary structure of HSA and BSA and reduced their aggregation close to their pI (Rawat and Bohidar, 2012). Akdogan et al. (Akdogan et al., 2011) demonstrated that [C₂MIm]BF₄ and 1-hexyl-3-methylimidazolium tetrafluoroborate ([C₆MIm]BF₄) from 15 to 50 % (v/v) caused HSA unfolding. Similarly, Sasmal and collaborators (Sasmal et al., 2011) showed that 1-pentyl-3-methylimidazolium chloride ([C₅MIm]Cl) from 0.3 to 1.5 M causes HSA denaturation. Interestingly, they also observed that adding 1.5 M of [C₅MIm]Cl to an HSA denatured by GuHCl, the IL helps with the protein refolding. Kumar et al. (Kumar Das et al., 2012) also observed HSA unfolding with the addition of 0.9 M of [C₅MIm]Br and a change in the structure and dynamics of HSA denatured by GuHCl. Hence, this set of studies reveals that lower concentrations of imidazolium ILs are more biocompatible to HSA than their concentrated solutions. Jo u rn al In addition to their concentration, the cationic nature and alkyl side chain length of imidazolium ILs also influence their effect on HSA structure. For instance, Silva et al. (Silva et al., 2014) evaluated the effect of altering the IL’s cation upon HSA stability, namely by studying solutions of 1-(2-hydroxyethyl)-3-methylimidazolium chloride ([OHC2MIm]Cl), 1-(2-methoxyethyl)-3-methylimidazolium chloride ([C₂OCMIm]Cl), [C₂MIm]Cl, [C₄MIm]Cl, [C₄MIm][N(CN)2] and 1-butyl-2,3dimethylimidazolium chloride ([C₄C1C1Im]Cl) at 0.005 M in water. Like BSA, the increase of the cation alkyl side chain length is associated with a higher destabilization of HSA. Moreover, it was also found that adding an alcohol or methoxy substituent to the cation reduces the destabilizing effect of the ILs on HSA. These researchers suggested that increasing the cation alkyl chain or the absence of hydrophilic substituents improves the IL-HSA surface of contact, hence increasing the protein unfolding. The hydrophobic interactions between IL and HSA are again predominant, although there is still a contribution from electrostatic interactions and hydrogen bonding. Furthermore, it is important to note that the nature of the anion (Cl− or [N(CN)2]−) modulated the cation-HSA binding. Most of the studies demonstrated that the cation usually dominates the HSAprotein interactions. Nonetheless, the anion can also interact with HSA or modulate the cation-HSA binding. For example, Akdogan and Hinderberger (Akdogan and Hinderberger, 2011) evaluated the impact of changing the anion in imidazolium-based ILs with short alkyl side chains upon HSA stability. At room temperature, 1-ethyl-3methyl imidazolium dicyanamide ([C₂MIm][N(CN)2]), 1-ethyl-3-methylimidazolium dimethylphosphate ([C₂MIm][Me₂PO₄]), 1-ethyl-3-methylimidazolium thiocyanate ([C₂MIm]SCN), 1-ethyl-3-methylimidazolium tetrafluoroborate ([C₂MIm]BF₄), 1-ethyl40 Journal Pre-proof 3-methyl imidazolium nitrate ([C₂MIm]NO₃) and [C₂MIm][EtSO₄] at 35 % (v/v) denatured HSA, similar to ethanolic aqueous solution (at 35% v/v). Interestingly, at low temperatures (-23.15 to 6.85 ºC), there was at least partial protein refolding. [C₂MIm][N(CN)2] led to the most unfolding in HSA structure, showing the anion has an impact on the intensity of the IL effects. Similarly, Page and co-workers (Page et al., 2009) observed a detrimental effect of the 1-butyl-3-methylimidazolium bis(trifluoromethylsulfonyl)imide ([C₄MIm][Tf₂N]), [C₄MIm]BF₄ and [C₄MIm]PF₆ on HSA structure. Specifically, neat ILs [98 % (v/v)] unfolded loop 1 of domain I of HSA when compared to the denaturing agents GuHCl and urea. On the other hand, in the presence of more diluted IL solutions [lower than 70 % (v/v) of ILs], there is a refolding of loop 1 from domain I in [C₄MIm]BF₄ (only one evaluated at lower concentration), with its recoupling with domains II and III. pr oo f For pyrrolidinium-based ILs, Kumari et al. (Kumari et al., 2014b) reported that [C4C1OPyrr]Br from 0.0167 to 0.1044 M unfolds HSA. As with other ILs, [C4C1OPyrr]Br interacts with HSA mainly by hydrophobic forces, followed by hydrogen bonding. The IL-protein interactions are spontaneous and entropy-driven and occur primarily at the hydrophobic pocket in domain IIA. Furthermore, these researchers confirmed that HSA increased the CMC of [C4C1OPyrr]Br in water, due to the IL-protein interactions. rn al Pr e- In summary, as shown for BSA, most ILs have disrupted the HSA structure, with the primary IL-HSA interaction being hydrophobic forces followed by hydrogen bonding (Akdogan et al., 2011; Akdogan and Hinderberger, 2011; Baker and Heller, 2009; Kumar Das et al., 2012; Kumari et al., 2014b; Page et al., 2009; Sasmal et al., 2011; Silva et al., 2014). The effect of cation was also dominant in HSA, particularly for ILs with longer cationic alkyl side chains (Akdogan and Hinderberger, 2011; Silva et al., 2014). Still, the anion also interacted with the protein though to a lesser extent, and mainly as a modulator of the cation effect (Akdogan and Hinderberger, 2011; Silva et al., 2014). Finally, the increase in IL concentration also enhanced the detrimental influence of ILs on BSA (Baker and Heller, 2009; Page et al., 2009). 3.2.2.5. β-Lactoglobulin (BLG) Jo u β-Lactoglobulin (BLG) is a globular protein from cow and other mammal's milk. BLG is from the class of lipocalins, and it can bind to different hydrophobic molecules and have a role in their transport (Kontopidis et al., 2004). Moreover, the food industry has a direct interest in BLG considering its properties can vary and affect the quality of dairy products. In this sense, BLG is a great and widely used model for food proteins (Barbiroli et al., 2022). As shown in Fig. 4.F, the BLG structure comprises a β-barrel with eight antiparallel β-strands, an α-helix with a 3-turn in the outside surface, and an additional β-strand (Kontopidis et al., 2004; Kuwata et al., 1999). This protein is hydrophilic and small, predominantly dimeric, but can dissociate to a monomer below pH 3 and aggregate into larger forms under many conditions (Crowther et al., 2016). Furthermore, BLG can form gels after denaturation and aggregation, having promising applications in material science (Loveday et al., 2017). BLG is a suitable model to study not only the interaction of ILs and food proteins but also has the potential for the development of novel BLG-ILs materials. Different research groups reported that specific ammonium-based ILs transform BLG secondary structure from a native β-barrel conformation into an α-helix form (Byrne et al., 2013; Takekiyo et al., 2013). For instance, Byrne et al. (Byrne et al., 2013) studied the influence of 20 to 80 wt% aqueous solutions of [N0,2,2,2][CH₃COO], 41 Journal Pre-proof pr oo f [N0,2,2,2]SO₄, triethylammonium trifluoroacetate ([N0,2,2,2][CF₃COO]) and [N0,2,2,2][CH₃SO₃] upon BLG structure. The basic [N0,2,2,2][CH₃COO] stabilized the native dimeric form of the protein at neutral and basic pH. On the other hand, the acidic [N0,2,2,2]SO₄ favored the monomeric conformation of BLG. Yet, both ILs preserved the β-barrel native structure of BLG. On the other hand, the IL with intermediate proton activity [N0,2,2,2][CF₃COO] caused the denaturation of BLG. [N0,2,2,2][CH₃SO₃] had the most intriguing results since at low concentrations (20 wt%), it preserved the native βbarrel protein structure and improved protein refolding after thermal denaturation; though at intermediary concentrations (40 wt%), there was a partial change in the secondary structure of the protein to α-helices and, after thermal denaturation, being observed the formation of amyloid fibrils (i.e., highly ordered structures formed after misfolding of specific proteins). At 80 wt% of [N0,2,2,2][CH₃SO₃], BLG structure was mainly comprised of α-helices and the protein maintained this form after thermal denaturation. Furthermore, the thermal stability of the BLG α-helix form was higher than its β-barrel native conformation. Takekiyo et al. (Takekiyo et al., 2013) also reported that the ammonium-based IL [N0,0,0,2]NO3 induces the formation of an α-helical structure for BLG by increasing the concentration from 15 to 50 mol%. This helical form for BLG behaved similarly to alcohol denaturation; however, the ILs generated a more disordered state in the transition from the β-barrel to the α-helix form. al Pr e- In addition to ammonium ILs, Takekiyo et al. (Takekiyo et al., 2014a, 2013) also reported that imidazolium-based ILs with nitrate anion can induce the formation of an α-helical BLG. [C₂MIm]NO₃ and [C₄MIm]NO₃ at 15 to 20 mol% favored the αhelical conformation of BLG, but different from the ammonium-based ILs, both nitrate ILs led to protein aggregation (Takekiyo et al., 2014a, 2013). When comparing [C₂MIm]NO₃ and [C₄MIm]NO₃, the first generated larger BLG aggregates, confirming a cation influence in the specific IL-protein interaction (Takekiyo et al., 2014a, 2013). Jo u rn Sankaranarayanan et al. (Sankaranarayanan et al., 2013) demonstrated that even diluted solutions (1 to 10 mM) of imidazolium-based ILs can generate a transition from a β-barrel structure to an α-helix form for BLG, being this pH-dependent. At acidic pH (pH 4) and in presence of [C₂MIm][EtSO₄], BLG goes from an initial helical structure into an intermediate β-turn form, followed by its native and more stable β-barrel conformation. Hence, there is a hierarchical transformation of BLG from its non-native state to its β-barrel form at acidic pH over time. However, at neutral pH (pH 7.5) and in presence of [C₂MIm][EtSO₄], the native β-barrel of BLG goes to a non-native helical form and returns to a β-barrel structure (non-hierarchical transformation). These researchers discovered that at pH 4, the protein was more surface-active with a higher interaction enthalpy, indicating a more pronounced contribution from solvation for the transition to BLG β-barrel form. On the other hand, the BLG transition from β-barrel to the non-native helical state at neutral pH was due to changes in the microviscosity of its environment caused by the addition of [C₂MIm][EtSO₄]. As can be seen from the different studies with ILs and BLG, the ILs induced conformational changes in the secondary structure of the protein from a β-barrel to a non-native α-helix form (Byrne et al., 2013; Sankaranarayanan et al., 2013; Takekiyo et al., 2014a, 2013). This phenomenon was dependent on the concentration of IL solutions and the medium pH (Byrne et al., 2013; Sankaranarayanan et al., 2013; Takekiyo et al., 2014a, 2013). Considering the variety of IL families and ILs concentrations with similar effects, specific changes in the microenvironment of the protein may favor the BLG αhelical form. 4. Final Remarks 42 Journal Pre-proof In this section, we will present the trends and knowledge gaps regarding the study of IL-protein interactions. Furthermore, we will try to find patterns associated with the effects of different classes and concentration ranges of ILs on proteins with distinct natures (hydrophobic and amphipathic, or hydrophilic). We will also discuss the opportunities for research and application of ILs in protein formulations. But before providing our view about IL-protein products, we need to state some aspects of ILs that should be properly addressed. Jo u rn al Pr e- pr oo f Ever since ILs emerged, these salts have been the focus of considerable attention due to their wide range of applications in several fields. This is only possible owing to the countless combinations of cations and anions composing the IL, giving rise to their designer solvents' character. However, this is also the reason behind some general misconceptions about ILs' biocompatibility, toxicity, biodegradability, and cost. Quite often, it is still possible to find recent studies describing ILs as stable organic salts with melting temperatures below 100 ºC, negligible vapor pressure, and a lack of flammability, hence being considered green solvents (Gomes et al., 2019; Kumar et al., 2017; Reslan and Kayser, 2018). All of these characteristics are true yet, not for all ILs, especially considering the numerous cation-anion possibilities known today. Therefore, one should be careful while presenting these universal statements. Firstly, there is a considerable amount of ILs that indeed have a melting point below 100 ºC; however, this is not one of the main criteria to determine if a salt is an IL or a traditional salt (e Silva et al., 2017; Greer et al., 2020). For instance, Freire et al. (e Silva et al., 2017) explored the differences between Coulombic-dominated salts and ILs based on their phase-forming abilities to create ABS as a function of temperature and demonstrated that a few known ILs would not have been considered ILs if the previous definition would prevail. Therefore, the interactions being established within the system and their magnitude have proven to be a more significant criterion to consider, thus suggesting that the difference between Coulombic-dominated salts and ILs goes beyond their melting temperature threshold of 100 ºC. Secondly, the low vapor pressure and nonflammability of ILs make these solvents better alternatives to the commonly employed volatile organic solvents, particularly considering the atmospheric pollution and the handler’s safety. In this sense, ILs can be considered greener solvents. Though it is also true that most of the first generation of ILs were not strictly green solvents as these were composed of imidazolium, pyridinium, and pyrrolidinium as the cation and hexafluorophosphate, tetrafluoroborate, and bis[(trifluoromethyl)sulfonyl]imide as some of the common anions. Due to the cation’s aromatic character and the anion’s hydrophobicity, these ILs presented low biocompatibility with biomolecules and a high terrestrial and aquatic toxicity (Cho et al., 2021; Greer et al., 2020). Besides, as these were the first ILs being synthesized, the costs were much higher. For these reasons, ILs are often still described as “toxic and expensive solvents”. Nevertheless, this is no longer the case, especially considering the ILs evolution, the preparation of second and third-generation ILs, as well as the improvements in their industrial production and commercialization. 4.1. Biocompatibility of ILs Driven by the need to develop more biocompatible and biodegradable ILs, researchers made use of the ILs designer solvent character by creating task-specific ILs according to the target application. Herein, ammonium-, betaine- and cholinium-based ILs emerged as attractive alternatives for the IL cation as well as the possibility of using amino acids, carboxylic acids, and lipids to create more biocompatible ILs (Ali et al., 2019; Gomes et al., 2019; Le Donne and Bodo, 2021; Mbakidi et al., 2021; Moshikur et 43 Journal Pre-proof al., 2020; Sivapragasam et al., 2019; Uddin et al., 2020). Among the ILs cations, cholinium is probably the most promising option as the derived ILs usually present low toxicity, a higher biodegradability rate, and a lower cost (Boethling et al., 2007; Kunz and Häckl, 2016; Pereira et al., 2016; Petkovic et al., 2010; Santos et al., 2015; Ventura et al., 2014). Cholinium-based ILs and salts have the additional advantage of being derived from vitamin B8, a quaternary ammonium cation, and some are even already used as nutritional supplements and pharmaceuticals (FEEDAP, 2011). Therefore, through a proper selection of cholinium as the cation and amino acids, carboxylic acids, or fatty acids as the anion, it is possible to create a fully bio-based IL that will simultaneously comply with the different hydrophobicity/hydrophilicity requirements of the target application and have an easy synthetic route. Pr e- pr oo f As fully bio-based ILs, the biocompatibility and biodegradability will no longer be a major concern, yet it is still patent that by increasing the alkyl side chain of the ILs components, there is higher cytotoxicity driven by the IL interaction with the organisms' lipidic membranes. Nevertheless, if the IL has a long enough alkyl side chain that can self-assemble and display a surface-active character, then there is a decrease in the toxicity since it decreases the possibility of direct interaction with the protection barriers of the organisms (Gomes et al., 2019; Gonçalves et al., 2021). It should be highlighted though, that for specific applications, namely antibacterial or antifungal uses, it is important that the IL presents an enhanced drug solubility and some cytotoxicity (Gonçalves et al., 2021; Moshikur et al., 2020; Sivapragasam et al., 2019; Wu et al., 2021). The former is one of the main reasons why ILs are being designed and used with active pharmaceutical ingredients (APIs). Jo u rn al APIs-based ILs are starting to revolutionize the pharmaceutical industry owing to their great potential for drug delivery by eliminating polymorphism, tailoring solubility, improving thermal stability, increasing dissolution, controlling drug release, modulating the surfactant properties, enhancing permeability of APIs, and modulating cytotoxicity on tumor cells, as recently reviewed by Wu et al. (Wu et al., 2021). Additionally, several other authors reviewed the biocompatibility of ILs for biological and pharmaceutical applications (Curreri et al., 2021; Gomes et al., 2019; Moshikur et al., 2020; Uddin et al., 2020), including the stabilization of enzymes and proteins (Ghorbanizamani and Timur, 2018; Kumar et al., 2017; Reslan and Kayser, 2018), which was deeply scrutinized in this review. Thus, this represents the importance of moving forward with the use of ILs not only as solvents but as stabilizing additives in protein and drug formulations, opening a new world of opportunities for ILs. 4.2. Perspective on the use ILs for the stabilization of protein-based bioproducts Firstly, we must reinforce that this discussion aims to guide future research to confirm the trends observed in this literature compilation, and not to be a definitive answer regarding IL-protein interactions. However, considering the overwhelming number of variables in this field, by dissecting the tendencies from over 100 studies, our goal was to shed light on the current knowledge gaps and opportunities in IL-protein research. To establish a more pragmatic analysis of the effects of ILs on non-enzymatic proteins, we evaluated the entries in Table 1 and Table 2 to obtain the amount of original ILs for every class (presented in Table 3) and the number of IL solutions that increased, maintained, or decreased protein stability according to the nature of the protein, range of IL concentration, and IL class (Table 4). Table 3. Amount of original ILs reported in Table 1 and Table 2 by IL class. 44 Journal Pre-proof IL class ILs (n) ILs (%) Ammonium 22 23.4 Cholinium 10 10.6 Guanidinium 2 2.1 Imidazolium 52 55.3 Phosphonium 1 1.1 Pyridinium and pyrrolidinium 7 7.4 94 100.0 f Total Pr e- pr oo Table 3 shows that there were 94 distinct ILs reported in Tables 1 and 2. The two IL classes with the greatest number of studies were the imidazolium-based ILs (with 52 different ILs) and ammonium-based ILs (with 22 entries). The other classes combined had 20 distinct ILs, with cholinium-based ILs with 10 entries, pyridiniumand pyrrolidinium-based ILs with 7, and only two and one distinct ILs for guanidinium and phosphonium families, respectively. Hence, the current IL-protein research is focused on two main classes (80 % of ILs studied), and there are still opportunities to unravel the effects of other IL classes. Regarding the specific effect of every class on proteins, Table 4 will explore this subject in detail. al Table 4. Amount of ionic liquid (IL) solutions from Table 1 and Table 2 that increase, maintain, or decrease non-enzymatic protein stability according to the protein nature (hydrophobic and amphipathic, or hydrophilic), range of IL concentrations (< 0.1 M, 0.1 to 1 M, or > 1 M), and IL class. Hydrophobic and amphipathic proteins 0.1 - 1 M >1M All ↑ = ↓ ↑ = ↓ ↑ = ↓ ↑ = ↓ Total Ammonium 0 0 0 5 0 0 5 0 4 10 0 4 14 Cholinium 2 1 9 0 0 0 0 0 0 2 1 9 12 Imidazolium 3 0 6 4 2 2 11 0 0 18 2 8 28 5 1 15 9 2 2 16 0 4 30 3 21 54 rn < 0.1 M Jo u IL class Total 21 13 20 54 54 < 0.1 M 0.1 - 1 M >1M All ↑ = ↓ ↑ = ↓ ↑ = ↓ ↑ = ↓ Total Ammonium 1 0 0 4 3 4 7 5 4 12 8 8 28 Cholinium 0 0 0 3 0 1 5 0 2 8 3 11 Hydrophilic proteins IL class 0 45 Journal Pre-proof Guanidinium 0 2 0 0 1 0 0 0 0 0 3 0 3 Imidazolium 9 4 31 7 9 18 4 2 30 20 15 79 114 Phosphonium 0 0 0 0 0 1 0 0 0 0 0 1 1 Pyridinium and pyrrolidinium 0 1 2 0 0 4 1 1 1 1 2 7 10 10 7 33 14 13 28 17 8 37 41 28 98 167 Total All proteins 50 55 62 167 167 71 68 82 221 221 Pr e- pr oo f As presented in Table 4, from 221 conditions, 41 IL solutions increased the stability of proteins, 31 maintained, and 119 decreased. There was diversity in the IL concentration range, with 71 very dilute solutions (lower than 0.1 M), 68 intermediary concentrations (0.1 to 1 M), and 82 concentrated IL solutions (above 1 M). For better visualization of the effect of ILs on protein stability, Fig. 5.A presents the impact of different concentrations of ILs, Fig. 5.B of IL classes, and Fig. 5.C and Fig. 5.D of concentration and protein type for ammonium and imidazolium ILs stabilization of proteins, respectively. We excluded the guanidinium and phosphonium classes from the analysis owing to their low number of entries (three and one, respectively). al Fig. 5. Percentage of IL solutions that maintain or increase non-enzymatic protein stability according to Table 4. The effect of ILs on proteins was divided into A) Concentration, B) IL class, C) Ammonium ILs (concentration and protein type), and D) Imidazolium ILs (concentration and protein type). Jo u rn Fig. 5.A suggests that all IL concentrations have the potential to enhance or maintain protein stability. However, IL concentrations above 0.1 M appear to be more biocompatible with proteins. It would be relevant to further investigate this phenomenon and confirm this trend, as this could help guide new research on this very broad topic. Furthermore, this Fig. 5.A also confirms the compatibility of ILs with proteins, with half of the solutions maintaining or increasing protein stability. For the IL classes in Fig. 5.B, the ammonium-based ILs had the most positive interactions with proteins, with 71.4 % preserving or enhancing their stability. Ammonium ILs were followed by cholinium-based ILs with 47.8 %, which are also regarded as biocompatible. However, even imidazolium, pyridinium, and pyrrolidinium ILs, often considered less compatible with biological systems, increased or maintained the stability of 38.7 and 30.0 % of the samples, respectively. Looking more specifically at the two most studied classes in Fig. 5.C, the increase of IL concentration above 1 M for ammonium ILs was negative for the stabilization of hydrophobic and amphipathic proteins, but slightly positive for hydrophilic proteins. In contrast, the increase in the concentration of imidazolium ILs was positive for the stabilization of hydrophobic and amphipathic proteins in Fig. 5.D, while the intermediary concentrations were more suitable for the hydrophilic. Another relevant piece of information from Fig. 5.B is the impact of the cation class on the effect of ILs on proteins. Many articles still cite that IL anions have a dominant effect on protein stabilization (Figueiredo et al., 2013; Jha et al., 2014; Klähn 46 Journal Pre-proof oo f et al., 2011; Patel et al., 2014; Yang, 2009; Yang et al., 2009). However, as extensively seen through this review, this phenomenon is not a consolidated fact. While there are articles evidencing a dominant influence of anions on certain proteins (Attri et al., 2014; Bui-Le et al., 2020; Fiebig et al., 2014; Fujita et al., 2007, 2006, 2005; Fujita and Ohno, 2010; Han et al., 2021), there are even more studies confirming the relevance of cations to directly interact with proteins or modulate the anion effect (Akdogan and Hinderberger, 2011; Geng et al., 2010, 2009; Huang et al., 2013; Jha et al., 2014; Jha and Venkatesu, 2016; Kumar and Venkatesu, 2014b; Kumari et al., 2014a; Shu et al., 2011; Silva et al., 2014; Singh et al., 2012; Takekiyo et al., 2014a, 2013; Veríssimo et al., 2021; Wang et al., 2012; Yan et al., 2012; Zhu et al., 2011). Furthermore, there is even a dominant effect of the cation on globular proteins such as BSA and HSA (Akdogan and Hinderberger, 2011; Geng et al., 2010, 2009; Kumari et al., 2014a; Shu et al., 2011; Silva et al., 2014; Singh et al., 2012). Hence, we question the current belief of an overall dominant effect of anions on proteins, as the IL-protein interactions will widely vary according to the IL class, protein, and medium conditions. Jo u rn al Pr e- pr The use of the Hofmeister series to explain the stabilization of proteins in ionic solutions is also a paradigm in dispute. For conventional salts, anions have a more pronounced agreement with the Hofmeister series than cations, with kosmotropic ions improving protein stability and chaotropic decreasing it (Zhang and Cremer, 2006). The Hofmeister series for anions have the following order, from kosmotropic to chaotropic: CO32-, SO42-, S2O32-, H2PO4-, F-, Cl-, Br-, NO3-, I-, ClO4 -, SCN- (Okur et al., 2017). For cations, the order from kosmotropic to chaotropic goes from NH 4+, K+, Na+, Li+, Mg2+, Ca2+, C(NH2)3+ (Okur et al., 2017). In addition to increasing protein stability, kosmotropic ions in general have higher surface tension and decrease the solubility of proteins (salting-out effect) (Zhang and Cremer, 2006). Furthermore, other physicalchemical and biological phenomena obey the Hofmeister series for conventional salts, such as enzyme activity, protein crystallization, optical rotation of sugar and amino acids, and bacterial growth. However, ILs do not usually behave as traditional salts. Kumar and Venkatesu (Kumar and Venkatesu, 2014c) addressed the impact of the Hofmeister series on the aptitude of ILs to stabilize proteins in a review on this topic. Authors concluded in their comprehensive literature analysis that this series is not suitable to explain or predict protein-IL interactions in most cases. They observed that the effect of ILs on proteins varies depending on the solvent environment (e.g., the concentration of IL and protein, temperature, co-solutes, pH), hence, just the nature of the ions will not always explain the complex interactions of ILs and these macromolecules. In another review regarding the association of the Hofmeister series and ILs for protein stabilization, Zhao (Zhao, 2016) concluded that diluted IL solutions overall agree with the Hofmeister series. However, concentrated or neat ILs have a distinct behavior, likely due to other factors like hydrogen-bond basicity, nucleophilicity, and hydrophobicity. Moreover, with an experimental approach, Paterová et al. (Paterová et al., 2013) demonstrated that changing the structure of peptides (i.e., uncapping their triglycine N-terminus) can reverse the Hofmeister trend of anions. Therefore, the protein functional groups also impact the IL-protein interactions. Due to the controversies regarding the use of the Hofmeister series to explain protein stabilization with ions, different groups have advocated for the use of the broader term “specific ion effects” to include the distinct properties and effects of ILs in addition to conventional salts (Okur et al., 2017; Paterová et al., 2013; Zhao, 2016). Thus, we can conclude that the Hofmeister series or even the nature of the cations and anions alone cannot explain or predict the effects of ILs on proteins, as the environmental conditions (e.g., pH, concentration, temperature, other solutes) and the 47 Journal Pre-proof proteins functional groups and structure will also play a role on the IL-protein interaction. Overall, it is clear that the IL class and concentration play an important role in the protein stabilization, especially considering the different types of protein stability. Therefore, we used the information presented in Table 1 and Table 2 to obtain the number of IL solutions that increased/maintained or decreased protein stability and correlated accordingly to the distinct effects. This data is presented in Table 5 and Fig. 6. f Table 5. Amount of IL solutions from Table 1 and Table 2 that increased/maintained or decreased the stability of non-enzymatic proteins, according to the type of stability (structural, thermal, activity, and aggregation), and IL class. * ↑ or = indicate ILs that improved protein aggregation (reduced aggregation) and ↓ represents ILs that increased protein aggregation. Pyridinium pyrrolidinium ↑ or = 2 1 0 0 19 oo Imidazolium ↑ or = ↓ 28 73 8 28 10 6 3 2 158 pr Cholinium ↑ or = ↓ 3 7 3 4 4 2 3 1 27 e- Ammonium ↑ or = ↓ Stability 16 16 Structural 14 11 Thermal 2 7 Activity 5 Aggregation* 7 78 Total ↓ 7 5 4 0 and All ↑ or = 49 26 16 13 282 Total ↓ 103 48 19 8 152 74 35 21 282 Pr IL Class rn al Fig. 6. Percentage of IL solutions that maintained or increased non-enzymatic proteins, according to Table 5, for the different types of protein stability parameters (structural, thermal, activity, and aggregation). For aggregation, improvement of stability includes ILs that decrease protein aggregation. Jo u As can be seen in Table 5 and Fig. 6, the effect of ILs on protein stability varies depending on the parameters evaluated. Here, the results changed when assessing the structure, thermal stability, activity, or aggregation rate of the proteins. Interestingly, the ILs effect on the structural and thermal stability of proteins was overall very similar, as shown in Fig. 6.A and Fig. 6.B. In general, 30 to 35 % of ILs increased or maintained the structural integrity and the thermal stability of proteins. Moreover, the results were also comparable considering the IL classes, with ammonium-based ILs having the best aptitude to preserve the structure and thermal stability of the proteins (50.0 and 56.0 %, respectively), followed by cholinium (30.0 and 42.9 %), imidazolium (27.7 and 22.2 %), and pyridinium and pyrrolidinium ILs (22.2 and 16.7 %). This phenomenon indicates the close relationship between the protein’s thermal stability and its structural integrity, as already suggested in literature (Bischof and He, 2006). As for the activity (Fig. 6.C), the ILs preserved 45.7 % of the protein's function (namely, Cyt C catalytic activity and GFP fluorescence activity). The higher ability of these proteins to preserve their activity when compared to their structural and thermal stability suggests that certain alterations to the protein do not compromise its function. Furthermore, it is evident that cholinium-based ILs are the most efficient in preserving the proteins’ stability (66.7 %), followed closely by the imidazolium ILs (62.5 %). However, ammonium and pyridinium/pyrrolidinium-based ILs impaired protein activity, preserving only 22.2 and 0.0 % of the samples’ function, respectively. 48 Journal Pre-proof Nonetheless, it must be noted that the sample size is low (n = 35) and includes the activity of only 2 proteins, hence, more studies are necessary to draw further conclusions for this parameter. The use of ILs to improve protein solubility is prevalent in literature (Fujita et al., 2005; Sivapragasam et al., 2016; Vasantha et al., 2014; Zhao et al., 2018), and its effectiveness can be confirmed in Fig. 6.D, with 61.9 % of ILs helping to reduce protein aggregation. For this parameter, the different IL classes performed similarly, with variations from 58.3 % for the ammonium ILs, 75.0 % for the cholinium, and 60.0 % for the imidazolium ILs. oo f Finally, to have a better understanding of the effects of the IL concentration and class on the proteins discussed in this review, Table 6 reports the amount of IL solutions that increased/maintained or decreased protein stability for each proteins. Furthermore, Fig. 7 presents the percentage of IL solutions that increased or preserved protein stability considering the IL concentration range whereas Fig. 8 displays these results for the different IL classes. 1 1 2 0 1 7 0 8 9 11 4 2 1 GFP 8 0 Mb 2 8 BSA 3 16 1 HSA 1 7 0 BLG 0 1 Tota 76 l 0 51 ↑ ↓ ↑ ↓ 1 3 1 6 0 4 3 47 Pyridinium and pyrrolidiniu m Imidazoliu Cholinium m ↑ ↓ ↑ ↓ ↑ ↓ ↑ 5 0 3 6 18 7 0 8 0 3 0 0 1 0 56 6 2 2 2 11 3 1 5 37 2 5 3 1 8 6 26 6 7 0 0 3 20 1 0 0 0 4 3 0 4 1 7 1 1 2 2 3 9 8 8 2 1 5 9 4 3 1 1 6 7 0 6 1 0 0 4 6 1 2 2 4 3 4 27 0 0 1 0 2 8 1 6 2 0 0 4 5 4 6 10 4 3 0 0 0 3 231 44 18 al Hb Cyt C ↓ rn ↑ ↓ ↑ Insul 8 12 10 in Tot Ammoniu al m Pr < 0.1 0.1 - 1 > 1 M M M All Jo u Prot ein e- pr Table 6. Amount of IL solutions from Table 1 and Table 2 that increase/maintain or decrease the stability of the non-enzymatic proteins, according to the different range of IL concentrations (< 0.1 M, 0.1 to 1 M, or > 1 M), and IL class. 0 104 231 125 All Tota l ↓ ↑ 2 0 6 ↓ 1 3 0 0 1 2 2 1 1 3 3 0 0 9 1 2 6 0 1 3 6 2 1 1 5 1 5 1 8 2 1 0 0 4 6 2 11 198 3 9 7 4 2 2 8 2 4 2 4 2 4 1 0 198 Fig. 7. Percentage of IL solutions that maintained or increased the stability of the non-enzymatic proteins, according to Table 6, for each protein and concentration range (< 0.1 M, 0.0 – 1 M, > 1 M, and all concentrations). Fig. 8. Percentage of IL solutions that maintained or increased the stability of the non-enzymatic proteins, according to Table 6, for each protein and IL class (ammonium-, cholinium-, imidazolium-, pyridiniumand pyrrolidinium-based ILs, and all IL classes). Table 6 and Fig. 7 show the effect of IL solutions on the stability of each proteins, considering three concentration ranges (i.e., < 0.1 M, 0.1 – 1 M, and > 1 M). For insulin, Cyt C, and GFP, the ILs improved or maintained the stability of more than 49 Journal Pre-proof oo f half of the samples (72.3, 50.0, and 64.9 %, respectively), while for Hb, BSA, and HSA, less than 20 % of ILs were able to preserve the proteins (12.5, 20.0, and 11.1 %, respectively). For Mb and BLG, around 40 % of the IL solutions preserved or increased the proteins’ stability. For insulin and Mb, the increase of IL concentration improved the protein stabilization, with the best results in the upper range for insulin (> 1 M) and the mid-range for Mb (0.1 - 1 M). However, the increase in the IL concentration impaired the stability of Cyt C, and GFP, which had the best results with diluted IL solutions (< 0.1 M). For Hb, BSA, and BLG, it is not possible to draw any conclusion regarding the concentration effect considering there were no samples above 1 M for BSA, only one sample below 1 M for BLG and between 0.1 and 1 M for Hb. For HSA, the ILs were highly detrimental to its stability (below 12.5 % stabilization) for all concentration ranges. Hence, the increase in IL concentration was positive for insulin and Mb stability, negative for Cyt C and GFP but had little effect on HSA. More studies for higher IL concentrations for BSA and lower for BLG and Hb are required for a trend to be observed. al Pr e- pr Table 6 also presents the impact of IL classes on the stability of the individual proteins, being the results in percentage shown in Fig. 8. The ammonium family was the best class for the stabilization of insulin, Cyt C, Mb, BSA, and BLG, while cholinium was the best for GFP and HSA. Although imidazolium-based ILs presented the highest stabilization for Hb, it was only 25.0 %, showing ILs tend to impair Hb stability. Overall, and as previously observed in Fig. 5, ammonium and cholinium ILs are the two most compatible classes with proteins. However, depending on the protein, they can be worse than imidazolium ILs for certain biomolecules (e.g., imidazolium ILs were better at stabilizing Hb than ammonium ILs; imidazolium ILs had a more positive effect on insulin than cholinium ILs). Therefore, it is possible to observe trends regarding the aptitude of an IL class to stabilize proteins, though, their effect varies depending on the protein. Jo u rn In this section, we managed to demonstrate the trends regarding the most studied IL classes and the effect of the IL concentration and class, as well as stability of each protein through different approaches. Overall, imidazolium and ammonium-based ILs dominate the studies for protein stabilization. Moreover, ammonium and choliniumbased ILs appear to be the most compatible classes with proteins, followed by imidazolium, pyridinium and pyrrolidinium ILs, considered the top four major IL families in this field. Additionally, concentrations of ILs above 0.1 M appear to be more biocompatible with proteins than very dilute aqueous IL solutions (below 0.1 M). Furthermore, ILs have a great aptitude to prevent protein aggregation (> 60 % of samples with decreased aggregation) and activity (around 50 %), including some IL families that are also adequate for the preservation of structural and thermal stability of proteins (30-35 %). We also discussed the trends regarding the best classes and concentration ranges for each protein in this review, highlighting the knowledge gaps that still prevent the determination of patterns for certain proteins. Finally, considering what was discussed regarding the biocompatibility of IL classes and the trends regarding the use of ILs for protein stabilization, the next section will use this information to dive into the strengths, weaknesses, opportunities, and threats of IL application in protein stabilization while using a SWOT (strengths, weaknesses, opportunities, and threats) analysis. 4.3. SWOT analysis 50 Journal Pre-proof This SWOT analysis was performed to elucidate the potential advantages and drawbacks of applying ILs for protein stabilization. Fig. 9 below presents the SWOT chart: Fig. 9. SWOT (strengths, weaknesses, opportunities, and threats) analysis of the ILs application for protein stabilization. oo f As can be seen in Fig. 9, the SWOT analysis provides an overview of the strengths, weaknesses, opportunities, and threats associated with the application of ILs for protein stabilization. For the strengths, the tunable and designed properties of ILs allow a diversity of physical-chemical properties and effects of ILs, which also encompass a wide hydrophilic-hydrophobic range of ILs and a variety of biocompatible ILs (Freire, 2016; Kunz and Häckl, 2016). There is also a vast amount of biocompatible ILs that can increase protein stability (Ghorbanizamani and Timur, 2018; Pereira et al., 2016). Furthermore, there are neat ILs and ILs solutions that can solubilize proteins that are not soluble in water (Schröder, 2017). al Pr e- pr These advantages generate different opportunities, including the application of ILs as additives to bioproducts (e.g., to increase protein stability) (Reslan and Kayser, 2018), biosensor modulators (e.g., to quench or dequench fluorescence of proteic biosensors (Veríssimo et al., 2021)), or for the development of formulations of biomaterials (e.g., encapsulation of proteins (Vieira et al., 2020), induction of formation of protein fibrils (Safavi and Farjami, 2010; Sankaranarayanan et al., 2012). Additionally, ILs can be used as alternative solvents to water and organic compounds, such as for the dissolution of proteins that are not water soluble or even as a nonaqueous solvent to improve certain enzymatic catalysis (Wakayama et al., 2019). Jo u rn As for the weaknesses, while there are classes as ammonium and cholinium ILs that usually include ILs with lower toxicity, there are still a lot of studies using families with toxic ILs, such as imidazolium, pyridinium and pyrrolidinium ILs (Cho et al., 2021; Gonçalves et al., 2021). Furthermore, as was seen in the last section in Fig. 5.B, around 50 % of the ILs impaired protein stability. While there are instances where protein denaturation may be wanted (e.g., protocols for protein denaturation), it can overall be a drawback for the application of a considerable proportion of IL solutions in bioproducts manufacturing/formulation. In this sense, there are also ILs that can alter the protein's native form or activity. Even though this effect can be sometimes considered positive (e.g., increase in enzyme activity, formation of protein fibrils for biomaterials), this can also impose additional difficulties in the application of ILs. There is also a weakness regarding the IL availability in the market, as the complexity of the synthesis of certain classes can increase their cost and limit their access (dos Santos et al., 2018; Wakayama et al., 2019). Hence, the current low availability or high prices of some specific ILs in the market is still a threat to their applications for protein stabilization. Another threat is the lack of approved ILs for human use by regulatory agencies, which would impose massive efforts to allow the application of ILs in pharmaceutical products (Hough and Rogers, 2007). Moreover, and as can be seen in this study, the same IL can have a completely different effect when the protein model is changed. This phenomenon impairs the viability of developing IL formulations for proteins, as it limits the application of each new IL formulation to distinct bioproducts. Finally, other biocompatible compounds can be used as protein stabilizers, such as polymers 51 Journal Pre-proof (Gombotz and Pettit, 1995) and carbohydrates (Colaco et al., 1994), with the advantage that many of them are already approved for human use. In summary, we offered a critical perspective regarding the use of ILs as protein stabilizers, highlighting the current lacunas in the field while guiding future studies to answer the existing queries and challenges. Most importantly, we hoped to evidence the interesting opportunities that ILs could bring not only to the development of proteinbased formulations and bio-products but also to biomaterials and biosensors. Author Contributions: Conceptualization: NVV and JFBP; investigation: NVV, FAV, and RCO; writing – original draft preparation: NVV and FAV; writing – review and editing: NVV, RCO, FAV, BL, RPSO, and JFBP; supervision: JFBP, RPSO, and BL. pr oo f Funding: The researchers acknowledge funding from São Paulo State Research Support Foundation – FAPESP (project 2019/15493-9, 2018/06908-8, 2018/25511-1), CNPq, and CAPES (001). CIEPQPF is supported by the Fundação para a Ciência e Tecnologia (FCT) through the projects UIDB/EQU/00102/2020 and UIDP/EQU/00102/2020. N.V. Veríssimo (2020/14144-8) and R. C. Oliveira (2020/10676-5) also acknowledge scholarship financial support from FAPESP. e- Acknowledgments: Some of the figures in this work were created with Bio Render.com. Pr Conflicts of Interest: The authors declare no conflict of interest. References Jo u rn al Adawiyah, N., Moniruzzaman, M., Hawatulaila, S., Goto, M., 2016. Ionic liquids as a potential tool for drug delivery systems. MedChemComm 7, 1881–1897. https://doi.org/10.1039/C6MD00358C Akdogan, Y., Hinderberger, D., 2011. Solvent-induced protein refolding at low temperatures. J. Phys. Chem. B 115, 15422–15429. https://doi.org/10.1021/jp209646f Akdogan, Y., Junk, M.J., Hinderberger, D., 2011. Effect of ionic liquids on the solution structure of human serum albumin. Biomacromolecules 12, 1072–1079. https://doi.org/10.1021/bm1014156 Alberts, B., Johnson, A., Lewis, J., Raff, M., Roberts, K., Walter, P., 2002. Analyzing protein structure and function, in: Molecular Biology of the Cell, 4th Ed. Garland Science, New York, USA. Ali, M.K., Moshikur, R.M., Wakabayashi, R., Tahara, Y., Moniruzzaman, M., Kamiya, N., Goto, M., 2019. Synthesis and characterization of choline–fatty-acid-based ionic liquids: A new biocompatible surfactant. J. Colloid Inter. Sci. 551, 72–80. https://doi.org/10.1016/j.jcis.2019.04.095 Artimo, P., Jonnalagedda, M., Arnold, K., Baratin, D., Csardi, G., de Castro, E., Duvaud, S., Flegel, V., Fortier, A., Gasteiger, E., Grosdidier, A., Hernandez, C., Ioannidis, V., Kuznetsov, D., Liechti, R., Moretti, S., Mostaguir, K., Redaschi, N., Rossier, G., Xenarios, I., Stockinger, H., 2012. ExPASy: SIB bioinformatics resource portal. Nucleic Acids Res. 40, W597–W603. http://dx.doi.org/10.1093/nar/gks400 52 Journal Pre-proof Jo u rn al Pr e- pr oo f Attri, P., Jha, I., Choi, E.H., Venkatesu, P., 2014. Variation in the structural changes of myoglobin in the presence of several protic ionic liquid. Int. J. Biol. Macromol. 69, 114–123. https://doi.org/10.1016/j.ijbiomac.2014.05.032 Bahreinipour, M., Zarei, H., Dashtestani, F., Rashidiani, J., Eskandari, K., Zarandi, S.A.M., Ardestani, S.K., Watabe, H., 2021. Radioprotective effect of nanoceria and magnetic flower-like iron oxide microparticles on gamma radiation-induced damage in BSA protein. AIMS Biophys. 8, 124–142. https://doi.org/10.3934/biophy.2021010 Baker, G.A., Heller, W.T., 2009. Small-angle neutron scattering studies of model protein denaturation in aqueous solutions of the ionic liquid 1-butyl-3methylimidazolium chloride. Chem. Eng. J. 147, 6–12. https://doi.org/10.1016/j.cej.2008.11.033 Baker, S.N., Zhao, H., Pandey, S., Heller, W.T., Bright, F.V., Baker, G.A., 2011. Fluorescence energy transfer efficiency in labeled yeast cytochrome C: A rapid screen for ion biocompatibility in aqueous ionic liquids. Phys. Chem. Chem. Phys. 13, 3642–3644. https://doi.org/10.1039/C0CP02345K Baldwin, R.L., 1996. How Hofmeister ion interactions affect protein stability. Biophys. J. 71, 2056–2063. https://doi.org/10.1016/S0006-3495(96)79404-3 Barbiroli, A., Iametti, S., Bonomi, F., 2022. Beta-lactoglobulin as a model food protein: How to promote, prevent, and exploit its unfolding processes. Molecules 27, 1131. https://doi.org/10.3390/molecules27031131 Belinskaia, D.A., Voronina, P.A., Batalova, A.A., Goncharov, N.V., 2021. Serum albumin. Encyclopedia 1, 65–75. https://doi.org/10.3390/encyclopedia1010009 Berman, H.M., Battistuz, T., Bhat, T.N., Bluhm, W.F., Bourne, P.E., Burkhardt, K., Feng, Z., Gilliland, G.L., Iype, L., Jain, S., 2002. The protein data bank. Acta Crystallogr. D 58, 899–907. https://doi.org/10.1107/S0907444902003451 Bertini, I., Cavallaro, G., Rosato, A., 2006. Cytochrome C: Occurrence and functions. Chem. Rev. 106, 90–115. https://doi.org/10.1021/cr050241v Bihari, M., Russell, T.P., Hoagland, D.A., 2010. Dissolution and dissolved state of cytochrome C in a neat, hydrophilic ionic liquid. Biomacromolecules 11, 2944– 2948. https://doi.org/10.1021/bm100735z Bischof, J.C., He, X., 2006. Thermal stability of proteins. Ann. NY Acad. Sci. 1066, 12–33. https://doi.org/10.1196/annals.1363.003 Boethling, R.S., Sommer, E., DiFiore, D., 2007. Designing small molecules for biodegradability. Chem. Rev. 107, 2207–2227. https://doi.org/10.1021/cr050952t Bowers, J., Butts, C.P., Martin, P.J., Vergara-Gutierrez, M.C., Heenan, R.K., 2004. Aggregation behavior of aqueous solutions of ionic liquids. Langmuir 20, 2191– 2198. https://doi.org/10.1021/la035940m Brange, J., Langkjœr, L., 1993. Insulin structure and stability, in: Stability and Characterization of Protein and Peptide Drugs. Plenum Press, New York, USA, pp. 315–350. Buettner, C.S., Cognigni, A., Schröder, C., Bica-Schröder, K., 2022. Surface-active ionic liquids: A review. J. Mol. Liq. 347, 118160. https://doi.org/10.1016/j.molliq.2021.118160 Bui-Le, L., Clarke, C.J., Bröhl, A., Brogan, A.P., Arpino, J.A., Polizzi, K.M., Hallett, J.P., 2020. Revealing the complexity of ionic liquid–protein interactions through a multi-technique investigation. Comm. Chem. 3, 1–9. https://doi.org/10.1038/s42004-020-0302-5 53 Journal Pre-proof Jo u rn al Pr e- pr oo f Butreddy, A., Janga, K.Y., Ajjarapu, S., Sarabu, S., Dudhipala, N., 2021. Instability of therapeutic proteins—An overview of stresses, stabilization mechanisms and analytical techniques involved in lyophilized proteins. Int. J. Biol. Macromol. 167, 309–325. https://doi.org/10.1016/j.ijbiomac.2020.11.188 Bye, J.W., Platts, L., Falconer, R.J., 2014. Biopharmaceutical liquid formulation: A review of the science of protein stability and solubility in aqueous environments. Biotechnol. Lett. 36, 869–875. https://doi.org/10.1007/s10529-013-1445-6 Byrne, N., Barrow, C., Mccluskey, A., 2013. Solvent induced changes in the conformational state of β-lactoglobulin and the influence of protic ionic liquids. J. Mol. Eng. Mater. 1, 1250004. https://doi.org/10.1142/S2251237312500049 Callahan, N., Tullman, J., Kelman, Z., Marino, J., 2020. Strategies for development of a next-generation protein sequencing platform. Trends Biochem. Sci. 45, 76–89. https://doi.org/10.1016/j.tibs.2019.09.005 Center for Biologics Evaluation and Research, 2020. Evaluating the safety and efficacy of hemoglobin-based blood substitutes [WWW Document]. FDA. URL https://www.fda.gov/vaccines-blood-biologics/biologics-researchprojects/evaluating-safety-and-efficacy-hemoglobin-based-blood-substitutes (accessed 2.23.22). Center for Drug Evaluation and Research, 2018. Information regarding insulin storage and switching between products in an emergency [WWW Document]. FDA. URL https://www.fda.gov/drugs/emergency-preparedness-drugs/informationregarding-insulin-storage-and-switching-between-products-emergency (accessed 2.21.22). Chen, J., Wang, Y., Zeng, Q., Ding, X., Huang, Y., 2014. Partition of proteins with extraction in aqueous two-phase system by hydroxyl ammonium-based ionic liquid. Anal. Methods 6, 4067–4076. https://doi.org/10.1039/C4AY00233D Cho, C.-W., Pham, T.P.T., Zhao, Y., Stolte, S., Yun, Y.-S., 2021. Review of the toxic effects of ionic liquids. Sci. Total Environ. 786, 147309. https://doi.org/10.1016/j.scitotenv.2021.147309 Chruszcz, M., Mikolajczak, K., Mank, N., Majorek, K.A., Porebski, P.J., Minor, W., 2013. Serum albumins—unusual allergens. BBA-Gen. Subjects 1830, 5375– 5381. https://doi.org/10.1016/j.bbagen.2013.06.016 Ciaccafava, A., Alberola, M., Hameury, S., Infossi, P., Giudici-Orticoni, M.-T., Lojou, E., 2011. Hydrogen bioelectrooxidation in ionic liquids: From cytochrome C3 redox behavior to hydrogenase activity. Electrochim. Acta 56, 3359–3368. https://doi.org/10.1016/j.electacta.2010.12.104 Colaco, C., Smith, C.J.S., Sen, S., Roser, D.H., Newman, Y., Ring, S., Roser, B.J., 1994. Chemistry of protein stabilization by trehalose, in: Formulation and Delivery of Proteins and Peptides, ACS Symposium Series. ACS Publications, Denver, USA, pp. 222–240. Crowther, J.M., Jameson, G.B., Hodgkinson, A.J., Dobson, R.C., 2016. Structure, oligomerisation and interactions of β-lactoglobulin, in: Milk Proteins-from Structure to Biological Properties and Health Aspects. InTech Open, Rijeka, Croatia. Cubitt, A.B., Heim, R., Adams, S.R., Boyd, A.E., Gross, L.A., Tsien, R.Y., 1995. Understanding, improving and using green fluorescent proteins. Trends Biochem. Sci. 20, 448–455. https://doi.org/10.1016/s0968-0004(00)89099-4 Curreri, A.M., Mitragotri, S., Tanner, E.E., 2021. Recent advances in ionic liquids in biomedicine. Adv. Sci. 8, 2004819. https://doi.org/10.1002/advs.202004819 54 Journal Pre-proof Jo u rn al Pr e- pr oo f Deller, M.C., Kong, L., Rupp, B., 2016. Protein stability: A crystallographer’s perspective. Acta Crystallogr. F 72, 72–95. https://doi.org/10.1107/S2053230X15024619 Ding, X., Wang, Y., Zeng, Q., Chen, J., Huang, Y., Xu, K., 2014. Design of functional guanidinium ionic liquid aqueous two-phase systems for the efficient purification of protein. Anal. Chim. Acta 815, 22–32. https://doi.org/10.1016/j.aca.2014.01.030 dos Santos, N.V., de Carvalho Santos-Ebinuma, V., Pessoa Junior, A., Pereira, J.F.B., 2018. Liquid-liquid extraction of biopharmaceuticals from fermented broth: Trends and future prospects. J. Chem. Technol. Biotechnol. 93, 1845–1863. https://doi.org/10.1002/jctb.5476 dos Santos, N.V., Saponi, C.F., Greaves, T.L., Pereira, J.F.B., 2019. Revealing a new fluorescence peak of the enhanced green fluorescent protein using threedimensional fluorescence spectroscopy. RSC Adv. 9, 22853–22858. https://doi.org/10.1039/C9RA02567G dos Santos, N.V., Saponi, C.F., Ryan, T.M., Primo, F.L., Greaves, T.L., Pereira, J.F.B., 2020. Reversible and irreversible fluorescence activity of the Enhanced Green Fluorescent Protein in pH: insights for the development of pH-biosensors. Int. J. Biol. Macromol. 164, 3474–3484. https://doi.org/10.1016/j.ijbiomac.2020.08.224 Du, Z., Yu, Y.-L., Wang, J.-H., 2007. Extraction of proteins from biological fluids by use of an Ionic liquid/aqueous two-phase system. Chem.-Euro. J. 13, 2130– 2137. https://doi.org/10.1002/chem.200601234 e Silva, F.A., Pereira, J.F., Kurnia, K.A., Ventura, S.P., Silva, A.M., Rogers, R.D., Coutinho, J.A., Freire, M.G., 2017. Temperature dependency of aqueous biphasic systems: An alternative approach for exploring the differences between Coulombic-dominated salts and ionic liquids. Chem. Comm. 53, 7298–7301. https://doi.org/10.1039/C7CC02294H Enoki, S., Saeki, K., Maki, K., Kuwajima, K., 2004. Acid denaturation and refolding of green fluorescent protein. Biochemistry 43, 14238–14248. https://doi.org/10.1021/bi048733+ Evens, R., Kaitin, K., 2015. The evolution of biotechnology and its impact on health care. Health Affairs 34, 210–219. https://doi.org/10.1377/hlthaff.2014.1023 ExPASy, 2018. ProtScale [WWW Document]. URL https://web.expasy.org/protscale (accessed 4.12.18). Fanali, G., Di Masi, A., Trezza, V., Marino, M., Fasano, M., Ascenzi, P., 2012. Human serum albumin: from bench to bedside. Mol. Aspects Med. 33, 209–290. https://doi.org/10.1016/j.mam.2011.12.002 FEEDAP, 2011. Scientific Opinion on safety and efficacy of choline chloride as a feed additive for all animal species. EFSA Journal 9, 2353. https://doi.org/10.2903/j.efsa.2011.2353 Ferrari, M., 2022. Outbreaks of Measles: Compounding Challenges in the DRC Democratic Republic of the Congo [WWW Document]. ReliefWeb. URL https://reliefweb.int/report/democratic-republic-congo/outbreaks-measlescompounding-challenges-drc (accessed 12.17.19). Fiebig, O.C., Mancini, E., Caputo, G., Vaden, T.D., 2014. Quantitative evaluation of myoglobin unfolding in the presence of guanidinium hydrochloride and ionic liquids in solution. J. Phys. Chem. B 118, 406–412. https://doi.org/10.1021/jp408061k 55 Journal Pre-proof Jo u rn al Pr e- pr oo f Figueiredo, A.M., Sardinha, J., Moore, G.R., Cabrita, E.J., 2013. Protein destabilisation in ionic liquids: The role of preferential interactions in denaturation. Phys. Chem. Chem. Phys. 15, 19632–19643. https://doi.org/DOI https://doi.org/10.1039/C3CP53395F Freire, M.G., 2016. Ionic-liquid-based Aqueous Biphasic Systems: Fundamentals and Applications, 1st ed. Springer-Verlag, Berlin, Germany. Fujita, K., Forsyth, M., MacFarlane, D.R., Reid, R.W., Elliott, G.D., 2006. Unexpected improvement in stability and utility of cytochrome C by solution in biocompatible ionic liquids. Biotechnol. Bioeng. 94, 1209–1213. https://doi.org/10.1002/bit.20928 Fujita, K., MacFarlane, D.R., Forsyth, M., 2005. Protein solubilising and stabilising ionic liquids. Chem. Comm. 4804–4806. https://doi.org/10.1039/B508238B Fujita, K., MacFarlane, D.R., Forsyth, M., Yoshizawa-Fujita, M., Murata, K., Nakamura, N., Ohno, H., 2007. Solubility and stability of cytochrome C in hydrated ionic liquids: Effect of oxo acid residues and kosmotropicity. Biomacromolecules 8, 2080–2086. https://doi.org/10.1021/bm070041o Fujita, K., Ohno, H., 2010. Enzymatic activity and thermal stability of metallo proteins in hydrated ionic liquids. Biopolymers 93, 1093–1099. https://doi.org/10.1002/bip.21526 Geng, F., Zheng, L., Liu, J., Yu, L., Tung, C., 2009. Interactions between a surface active imidazolium ionic liquid and BSA. Colloid Polym. Sci. 287, 1253–1259. https://doi.org/10.1007/s00396-009-2085-1 Geng, F., Zheng, L., Yu, L., Li, G., Tung, C., 2010. Interaction of bovine serum albumin and long-chain imidazolium ionic liquid measured by fluorescence spectra and surface tension. Process Biochem. 45, 306–311. https://doi.org/10.1016/j.procbio.2009.10.001 Ghorbanizamani, F., Timur, S., 2018. Ionic liquids from biocompatibility and rlectrochemical aspects toward applying in biosensing devices. Anal. Chem. 90, 640–648. https://doi.org/10.1021/acs.analchem.7b03596 Giardina, B., Messana, I., Scatena, R., Castagnola, M., 1995. The multiple functions of hemoglobin. Crit. Rev. Biochem. Mol. 30, 165–196. https://doi.org/10.3109/10409239509085142 Gombotz, W.R., Pettit, D.K., 1995. Biodegradable polymers for protein and peptide drug delivery. Bioconjugate Chem. 6, 332–351. https://doi.org/10.1021/bc00034a002 Gomes, J.M., Silva, S.S., Reis, R.L., 2019. Biocompatible ionic liquids: Fundamental behaviours and applications. Chem. Soc. Rev. 48, 4317–4335. https://doi.org/10.1039/C9CS00016J Gonçalves, A.R., Paredes, X., Cristino, A.F., Santos, F.J.V., Queirós, C.S., 2021. Ionic liquids—A review of their toxicity to living organisms. Int. J. Mol. Sci. 22, 5612. https://doi.org/10.3390/ijms22115612 Greenfield, N.J., 2006. Using circular dichroism spectra to estimate protein secondary structure. Nat. Protoc. 1, 2876–2890. https://doi.org/10.1038/nprot.2006.202 Greer, A.J., Jacquemin, J., Hardacre, C., 2020. Industrial applications of ionic liquids. Molecules 25, 5207. https://doi.org/10.3390/molecules25215207 Guncheva, M., Ossowicz, P., Janus, E., Todinova, S., Yancheva, D., 2019. Elucidation of the effect of some cholinium amino acid ionic liquids on the thermal and the conformational stability of insulin. J. Mol. Liq. 283, 257–262. https://doi.org/10.1016/j.molliq.2019.03.074 56 Journal Pre-proof Jo u rn al Pr e- pr oo f Guruprasad, K., Reddy, B.B., Pandit, M.W., 1990. Correlation between stability of a protein and its dipeptide composition: A novel approach for predicting in vivo stability of a protein from its primary sequence. Protein Eng. Des. Sel. 4, 155– 161. https://doi.org/10.1093/protein/4.2.155 Han, Q., Ryan, T.M., Rosado, C.J., Drummond, C.J., Greaves, T.L., 2021. Effect of ionic liquids on the fluorescence properties and aggregation of superfolder green fluorescence protein. J. Colloid Inter. Sci. 591, 96–105. https://doi.org/10.1016/j.jcis.2021.02.002 Hargrove, M.S., Krzywda, S., Wilkinson, A.J., Dou, Y., Ikeda-Saito, M., Olson, J.S., 1994. Stability of myoglobin: A model for the folding of heme proteins. Biochemistry 33, 11767–11775. https://doi.org/10.1021/bi00205a012 Heller, W.T., O’Neill, H.M., Zhang, Q., Baker, G.A., 2010. Characterization of the influence of the ionic liquid 1-butyl-3-methylimidazolium chloride on the structure and thermal stability of green fluorescent protein. J. Phys. Chem. B 114, 13866–13871. https://doi.org/10.1021/jp105611b Ho, B., Baryshnikova, A., Brown, G.W., 2018. Unification of protein abundance datasets yields a quantitative Saccharomyces cerevisiae proteome. Cell Syst. 6, 192-205. e3. https://doi.org/10.1016/j.cels.2017.12.004 Hong, H., Joh, N.H., Bowie, J.U., Tamm, L.K., 2009. Methods for measuring the thermodynamic stability of membrane proteins. Method. Enzymol. 455, 213– 236. https://doi.org/10.1016/S0076-6879(08)04208-0 Hough, W.L., Rogers, R.D., 2007. Ionic liquids then and now: From solvents to materials to active pharmaceutical ingredients. B. Chem. Soc. JPN 80, 2262– 2269. https://doi.org/10.1246/bcsj.80.2262 Huang, C., Bai, H., Li, C., Shi, G., 2011. A graphene oxide/hemoglobin composite hydrogel for enzymatic catalysis in organic solvents. Chem. Comm. 47, 4962– 4964. https://doi.org/10.1039/C1CC10412H Huang, R., Zhang, S., Pan, L., Li, J., Liu, F., Liu, H., 2013. Spectroscopic studies on the interactions between imidazolium chloride ionic liquids and bovine serum albumin. Spectrochim. Acta A 104, 377–382. https://doi.org/10.1016/j.saa.2012.11.087 Hubbard, S.R., Hendrickson, W.A., Lambright, D.G., Boxer, S.G., 1990. X-ray crystal structure of a recombinant human myoglobin mutant at 2· 8 Å resolution. J. Mol. Biol. 213, 215–218. https://doi.org/10.1016/S0022-2836(05)80181-0 Humphreys, G., 2011. Vaccination: rattling the supply chain. B. World Health Organ. 89, 324. https://doi.org/10.2471/BLT.11.030511 Ilari, A., Savino, C., 2008. Protein structure determination by X-ray crystallography. Bioinformatics 63–87. https://doi.org/10.1007/978-1-60327-159-2_3 Iyer, P.V., Ananthanarayan, L., 2008. Enzyme stability and stabilization—Aqueous and non-aqueous environment. Process Biochem. 43, 1019–1032. https://doi.org/10.1016/j.procbio.2008.06.004 Jaganathan, M., Ramakrishnan, C., Velmurugan, D., Dhathathreyan, A., 2015. Understanding ethylammonium nitrate stabilized cytochrome C–Molecular dynamics and experimental approach. J. Mol. Strut. 1081, 334–341. https://doi.org/10.1016/j.molstruc.2014.10.049 Jahanban-Esfahlan, A., Ostadrahimi, A., Jahanban-Esfahlan, R., Roufegarinejad, L., Tabibiazar, M., Amarowicz, R., 2019. Recent developments in the detection of bovine serum albumin. Int. J. Biol. Macromol. 138, 602–617. https://doi.org/10.1016/j.ijbiomac.2019.07.096 57 Journal Pre-proof Jo u rn al Pr e- pr oo f Jha, I., Attri, P., Venkatesu, P., 2014. Unexpected effects of the alteration of structure and stability of myoglobin and hemoglobin in ammonium-based ionic liquids. Phys. Chem. Chem. Phys. 16, 5514–5526. https://doi.org/10.1039/C3CP54398F Jha, I., Venkatesu, P., 2016. Unprecedented improvement in the stability of hemoglobin in the presence of promising green solvent 1-allyl-3-methylimidazolium chloride. ACS Sustain. Chem. Eng. 4, 413–421. https://doi.org/10.1021/acssuschemeng.5b00939 Kikhney, A.G., Svergun, D.I., 2015. A practical guide to small angle X-ray scattering (SAXS) of flexible and intrinsically disordered proteins. FEBS Lett. 589, 2570– 2577. https://doi.org/10.1016/j.febslet.2015.08.027 Klähn, M., Lim, G.S., Wu, P., 2011. How ion properties determine the stability of a lipase enzyme in ionic liquids: A molecular dynamics study. Phys. Chem. Chem. Phys. 13, 18647–18660. https://doi.org/10.1039/C1CP22056J Kong, J., Yu, S., 2007. Fourier transform infrared spectroscopic analysis of protein secondary structures. Acta Bioch. Bioph. Sin. 39, 549–559. https://doi.org/10.1111/j.1745-7270.2007.00320.x C Kontopidis, G., Holt, C., Sawyer, L., 2004. Invited review: β-lactoglobulin: binding properties, structure, and function. J. Dairy Sci. 87, 785–796. https://doi.org/10.3168/jds.S0022-0302(04)73222-1 Kouchakzadeh, H., Shojaosadati, S.A., Shokri, F., 2014. Efficient loading and entrapment of tamoxifen in human serum albumin based nanoparticulate delivery system by a modified desolvation technique. Chem. Eng. Res. Des. 92, 1681–1692. https://doi.org/10.1016/j.cherd.2013.11.024 Kumar, A., Bisht, M., Venkatesu, P., 2017. Biocompatibility of ionic liquids towards protein stability: A comprehensive overview on the current understanding and their implications. Int. J. Biol. Macromol. 96, 611–651. https://doi.org/10.1016/j.ijbiomac.2016.12.005 Kumar, A., Venkatesu, P., 2014a. The stability of insulin in the presence of short alkyl chain imidazolium-based ionic liquids. RSC Adv. 4, 4487–4499. https://doi.org/10.1039/C3RA44477E Kumar, A., Venkatesu, P., 2014b. A comparative study of myoglobin stability in the presence of Hofmeister anions of ionic liquids and ionic salts. Process Biochem. 49, 2158–2169. https://doi.org/10.1016/j.procbio.2014.09.014 Kumar, A., Venkatesu, P., 2014c. Does the stability of proteins in ionic liquids obey the Hofmeister series? Int. J. Biol. Macromol. 63, 244–253. https://doi.org/10.1016/j.ijbiomac.2013.10.031 Kumar, A., Venkatesu, P., 2013. Prevention of insulin self-aggregation by a protic ionic liquid. RSC Adv. 3, 362–367. https://doi.org/10.1039/C2RA22277A Kumar Das, D., Kumar Das, A., Kumar Mandal, A., Mondal, T., Bhattacharyya, K., 2012. Effect of an ionic liquid on the unfolding of human serum albumin: A fluorescence correlation spectroscopy study. ChemPhysChem 13, 1949–1955. https://doi.org/10.1002/cphc.201100421 Kumari, M., Maurya, J.K., Singh, U.K., Khan, A.B., Ali, M., Singh, P., Patel, R., 2014a. Spectroscopic and docking studies on the interaction between pyrrolidinium based ionic liquid and bovine serum albumin. Spectrochim. Acta A 124, 349– 356. https://doi.org/10.1016/j.saa.2014.01.012 Kumari, M., Maurya, J.K., Tasleem, M., Singh, P., Patel, R., 2014b. Probing HSA-ionic liquid interactions by spectroscopic and molecular docking methods. J. Photoch. Photobio. B 138, 27–35. https://doi.org/10.1016/j.jphotobiol.2014.05.009 58 Journal Pre-proof Jo u rn al Pr e- pr oo f Kunz, W., Häckl, K., 2016. The hype with ionic liquids as solvents. Chem. Phys. Lett. 661, 6–12. https://doi.org/10.1016/j.cplett.2016.07.044 Kuwata, K., Hoshino, M., Forge, V., Era, S., Batt, C.A., Goto, Y., 1999. Solution structure and dynamics of bovine β-lactoglobulin A. Protein Sci. 8, 2541–2545. https://doi.org/10.1110/ps.8.11.2541 Lambert, T.J., 2019. FPbase: A community-editable fluorescent protein database. Nat. Methods 16, 277–278. https://doi.org/10.1038/s41592-019-0352-8 Le Donne, A., Bodo, E., 2021. Cholinium amino acid-based ionic liquids. Biophys. Rev. 13, 147–160. https://doi.org/10.1007/s12551-021-00782-0 Lee, J., Freddolino, P.L., Zhang, Y., 2017. Ab initio protein structure prediction, in: From Protein Structure to Function with Bioinformatics. Springer, Dordrecht, Netherlands, pp. 3–35. Li, D., Gao, Y., Pan, X., Wei, D., Guo, B., Yang, C., Liu, B., 2019. MD and DSC study of bioactive structural stability of insulin in various imidazolium ionic liquids. J. Mol. Liq. 277, 971–976. https://doi.org/10.1016/j.molliq.2019.01.039 Li, W., Wu, P., 2014. Insights into the denaturation of bovine serum albumin with a thermo-responsive ionic liquid. Soft Matter 10, 6161–6171. https://doi.org/10.1039/C4SM00941J Lin, X., Wang, Y., Zeng, Q., Ding, X., Chen, J., 2013. Extraction and separation of proteins by ionic liquid aqueous two-phase system. Analyst 138, 6445–6453. https://doi.org/10.1039/C3AN01301D Liu, H.-L., Hsu, J.-P., 2005. Recent developments in structural proteomics for protein structure determination. Proteomics 5, 2056–2068. https://doi.org/10.1002/pmic.200401104 Loveday, S.M., Anema, S.G., Singh, H., 2017. β-Lactoglobulin nanofibrils: The long and the short of it. Int. Dairy J. 67, 35–45. https://doi.org/10.1016/j.idairyj.2016.09.011 Lukin, J.A., Kontaxis, G., Simplaceanu, V., Yuan, Y., Bax, A., Ho, C., 2003. Quaternary structure of hemoglobin in solution. P. Natl. A. Sci. USA 100, 517– 520. https://doi.org/10.1073/pnas.232715799 Ma, G.J., Ferhan, A.R., Jackman, J.A., Cho, N.-J., 2020. Conformational flexibility of fatty acid-free bovine serum albumin proteins enables superior antifouling coatings. Commun. Mater. 1, 1–11. https://doi.org/10.1038/s43246-020-0047-9 Maier, R., Fries, M.R., Buchholz, C., Zhang, F., Schreiber, F., 2021. Human versus bovine serum albumin: A subtle difference in hydrophobicity leads to large differences in bulk and interface behavior. Cryst. Growth Des. 21, 5451–5459. https://doi.org/10.1021/acs.cgd.1c00730 Majorek, K.A., Porebski, P.J., Dayal, A., Zimmerman, M.D., Jablonska, K., Stewart, A.J., Chruszcz, M., Minor, W., 2012. Structural and immunologic characterization of bovine, horse, and rabbit serum albumins. Mol. Immunol. 52, 174–182. https://doi.org/10.1016/j.molimm.2012.05.011 Manning, M.C., Chou, D.K., Murphy, B.M., Payne, R.W., Katayama, D.S., 2010. Stability of protein pharmaceuticals: An update. Pharm. Res. 27, 544–575. https://doi.org/10.1007/s11095-009-0045-6 Manning, M.C., Patel, K., Borchardt, R.T., 1989. Stability of protein pharmaceuticals. Pharm. Res. 6, 903–918. https://doi.org/10.1023/A:1015929109894 Marengo-Rowe, A.J., 2006. Structure-function relations of human hemoglobins. Baylor Univ. Med. Cent. P. 19, 239–245. https://doi.org/10.1080/08998280.2006.11928171 59 Journal Pre-proof Jo u rn al Pr e- pr oo f Mayer, J.P., Zhang, F., DiMarchi, R.D., 2007. Insulin structure and function. Peptide Sci. 88, 687–713. https://doi.org/10.1002/bip.20734 Mazzola, P.G., Ishii, M., Chau, E., Cholewa, O., Penna, T.C., 2006. Stability of green fluorescent protein (GFP) in chlorine solutions of varying pH. Biotechnol. Progr. 22, 1702–1707. https://doi.org/10.1021/bp060217i Mbakidi, J.-P., Barjhoux, I., Aguibi, K., Geffard, A., Rioult, D., Palos Ladeiro, M., Bouquillon, S., 2021. Synthesis of new betaine-based ionic liquids by using a “one-pot” amidation process and evaluation of their ecotoxicity through a new method involving a hemocyte-based bioassay. ACS Sustain. Chem. Eng. 9, 15427–15441. https://doi.org/10.1021/acssuschemeng.1c03982 McManus, J.J., Charbonneau, P., Zaccarelli, E., Asherie, N., 2016. The physics of protein self-assembly. Curr. Opin. Colloid. In. 22, 73–79. https://doi.org/10.1016/j.cocis.2016.02.011 Milne, J.L., Borgnia, M.J., Bartesaghi, A., Tran, E.E., Earl, L.A., Schauder, D.M., Lengyel, J., Pierson, J., Patwardhan, A., Subramaniam, S., 2013. Cryo‐electron microscopy–A primer for the non‐microscopist. FEBS J. 280, 28–45. https://doi.org/10.1111/febs.12078 Miskolczy, Z., Sebők-Nagy, K., Biczók, L., Göktürk, S., 2004. Aggregation and micelle formation of ionic liquids in aqueous solution. Chem. Phys. Lett. 400, 296–300. https://doi.org/10.1016/j.cplett.2004.10.127 Mongan, J., Case, D.A., 2005. Biomolecular simulations at constant pH. Curr. Opin. Struc. Biol. 15, 157–163. https://doi.org/10.1016/j.sbi.2005.02.002 Moniruzzaman, M., Kamiya, N., Nakashima, K., Goto, M., 2008. Formation of reverse micelles in a room‐temperature ionic liquid. ChemPhysChem 9, 689–692. https://doi.org/10.1002/cphc.200700802 Moshikur, R.M., Chowdhury, M.R., Moniruzzaman, M., Goto, M., 2020. Biocompatible ionic liquids and their applications in pharmaceutics. Green Chem. 22, 8116–8139. https://doi.org/10.1039/D0GC02387F Nelson, D.L., Cox, M.M., 2012. Lehninger principles of biochemistry, Sixth Ed. WH Freeman and Company, New York, USA. Noritomi, H., Minamisawa, K., Kamiya, R., Kato, S., 2011. Thermal stability of proteins in the presence of aprotic ionic liquids. J. Biomed. Sci. Eng. 4, 94. https://doi.org/10.4236/jbise.2011.42013 Ohtake, S., Kita, Y., Arakawa, T., 2011. Interactions of formulation excipients with proteins in solution and in the dried state. Adv. Drug Deliver. Rev. 63, 1053– 1073. https://doi.org/10.1016/j.addr.2011.06.011 Okur, H.I., Hladílková, J., Rembert, K.B., Cho, Y., Heyda, J., Dzubiella, J., Cremer, P.S., Jungwirth, P., 2017. Beyond the Hofmeister series: Ion-specific effects on proteins and their biological functions. J. Phys. Chem. B 121, 1997–2014. https://doi.org/10.1021/acs.jpcb.6b10797 Oliveira, V.C. de, Serrano Gallardo, P., Bezerra Cavalcante, R., Arcêncio, R.A., Pinto, I.C., 2015. Weaknesses of vaccine storage in primary healthcare centers. Rev. Bras. Enferm. https://doi.org/10.1590/0034-7167.2015680215i Ordway, G.A., Garry, D.J., 2004. Myoglobin: An essential hemoprotein in striated muscle. J. Exp. Biol. 207, 3441–3446. https://doi.org/10.1242/jeb.01172 Ow, Y.-L.P., Green, D.R., Hao, Z., Mak, T.W., 2008. Cytochrome C: Functions beyond respiration. Nat. Rev. Mol. Cell Biol. 9, 532–542. https://doi.org/10.1038/nrm2434 60 Journal Pre-proof Jo u rn al Pr e- pr oo f Page, T.A., Kraut, N.D., Page, P.M., Baker, G.A., Bright, F.V., 2009. Dynamics of loop 1 of domain I in human serum albumin when dissolved in ionic liquids. J. Phys. Chem. B 113, 12825–12830. https://doi.org/10.1021/jp904475v Patel, J., Kothari, R., Tunga, R., Ritter, N., Tunga, B., 2011. Stability considerations for biopharmaceuticals: Overview of protein and peptide degradation pathways. Bioprocess Int. 9, 2–11. Patel, R., Kumari, M., Khan, A.B., 2014. Recent advances in the applications of ionic liquids in protein stability and activity: A review. Appl. Biochem. Biotechnol. 172, 3701–3720. https://doi.org/10.1007/s12010-014-0813-6 Paterová, J., Rembert, K.B., Heyda, J., Kurra, Y., Okur, H.I., Liu, W.R., Hilty, C., Cremer, P.S., Jungwirth, P., 2013. Reversal of the Hofmeister series: Specific ion effects on peptides. J. Phys. Chem. B 117, 8150–8158. https://doi.org/10.1021/jp405683s Pereira, J.F., Deutschmann, R., Rogers, R.D., 2016. On the hunt for more benign and biocompatible ABS, in: Ionic-Liquid-Based Aqueous Biphasic Systems. Springer-Verlag, Berlin, Germany, pp. 247–284. Peters Jr, T., 1995. All about albumin: Biochemistry, genetics, and medical applications. Academic press, New York, USA. Petkovic, M., Ferguson, J.L., Gunaratne, H.N., Ferreira, R., Leitao, M.C., Seddon, K.R., Rebelo, L.P.N., Pereira, C.S., 2010. Novel biocompatible cholinium-based ionic liquids—Toxicity and biodegradability. Green Chem. 12, 643–649. https://doi.org/10.1039/B922247B Petoukhov, M.V., Svergun, D.I., 2007. Analysis of X-ray and neutron scattering from biomacromolecular solutions. Curr. Opin. Struc. Biol. 17, 562–571. https://doi.org/10.1016/j.sbi.2007.06.009 Pettersen, E.F., Goddard, T.D., Huang, C.C., Couch, G.S., Greenblatt, D.M., Meng, E.C., Ferrin, T.E., 2004. UCSF Chimera—A visualization system for exploratory research and analysis. J. Comput. Chem. 25, 1605–1612. https://doi.org/10.1002/jcc.20084 Ponomarenko, E.A., Poverennaya, E.V., Ilgisonis, E.V., Pyatnitskiy, M.A., Kopylov, A.T., Zgoda, V.G., Lisitsa, A.V., Archakov, A.I., 2016. The size of the human proteome: The width and depth. Int. J. Anal. Chem. 2016. https://doi.org/10.1155/2016/7436849 Prospec, 2022. Bovine serum albumin (BSA) [WWW Document]. Prospecbio. URL https://www.prospecbio.com/bovine_serum_albumin (accessed 3.13.22). Prymula, R., Simko, R., Povey, M., Kulcsar, A., 2016. Varicella vaccine without human serum albumin versus licensed varicella vaccine in children during the second year of life: a randomized, double-blind, non-inferiority trial. BMC Pediatr. 16, 1–7. https://doi.org/10.1186/s12887-016-0546-5 Rawat, K., Bohidar, H.B., 2015. Heparin-like native protein aggregate dissociation by 1-alkyl-3-methyl imidazolium chloride ionic liquids. Int. J. Biol. Macromol. 73, 23–30. https://doi.org/10.1016/j.ijbiomac.2014.10.057 Rawat, K., Bohidar, H.B., 2012. Universal charge quenching and stability of proteins in 1-methyl-3-alkyl (hexyl/octyl) imidazolium chloride ionic liquid solutions. J. Phys. Chem. B 116, 11065–11074. https://doi.org/10.1021/jp3049108 Reslan, M., Kayser, V., 2018. Ionic liquids as biocompatible stabilizers of proteins. Biophys. Rev. 10, 781–793. https://doi.org/10.1007/s12551-018-0407-6 Safavi, A., Farjami, F., 2010. Hydrogen peroxide biosensor based on a myoglobin/hydrophilic room temperature ionic liquid film. Anal. Biochem. 402, 20–25. https://doi.org/10.1016/j.ab.2010.03.013 61 Journal Pre-proof Jo u rn al Pr e- pr oo f Sankaranarayanan, K., Sathyaraj, G., Nair, B.U., Dhathathreyan, A., 2012. Reversible and irreversible conformational transitions in myoglobin: Role of hydrated amino acid ionic liquid. J. Phys. Chem. B 116, 4175–4180. https://doi.org/10.1021/jp300596z Sankaranarayanan, K., Sreedhar, B., Nair, B.U., Dhathathreyan, A., 2013. Microviscosity-induced conformational transition in β-lactoglobulin in the presence of an ionic liquid. J. Phys. Chem. B 117, 1234–1240. https://doi.org/10.1021/jp310198f Santos, C.A. dos, Mazzola, P.G., Silva, P.H.S., Cholewa, O., Penna, T.C.V., 2007. Preliminary study on the potential utility of GFP as a biosensor for drug stability in parenteral solutions. Biotechnol. Progress 23, 979–984. https://doi.org/10.1021/bp070090c Santos, J., Gonçalves, A., Pereira, J., Figueiredo, B., e Silva, F., Coutinho, J., Ventura, S., Goncalves, F., 2015. Environmental safety of cholinium-based ionic liquids: Assessing structure–ecotoxicity relationships. Green Chem. 17, 4657–4668. https://doi.org/10.1039/C5GC01129A Sasmal, D.K., Mondal, T., Sen Mojumdar, S., Choudhury, A., Banerjee, R., Bhattacharyya, K., 2011. An FCS study of unfolding and refolding of CPMlabeled human serum albumin: role of ionic liquid. J. Phys. Chem. B 115, 13075–13083. https://doi.org/10.1021/jp207829y Schofield, T.L., 2009. Vaccine stability study design and analysis to support product licensure. Biologicals 37, 387–396. https://doi.org/10.1016/j.biologicals.2009.08.009 Schröder, C., 2017. Proteins in ionic liquids: Current status of experiments and simulations, in: Ionic Liquids II. Springer, Cham, Switzerland, pp. 127–152. Sedlák, E., Stagg, L., Wittung-Stafshede, P., 2008. Effect of Hofmeister ions on protein thermal stability: Roles of ion hydration and peptide groups? Arch. Biochem. Biophys. 479, 69–73. https://doi.org/10.1016/j.abb.2008.08.013 Sen Mojumdar, S., Chowdhury, R., Chattoraj, S., Bhattacharyya, K., 2012. Role of ionic liquid on the conformational dynamics in the native, molten globule, and unfolded states of cytochrome C: A fluorescence correlation spectroscopy study. J. Phys. Chem. B 116, 12189–12198. https://doi.org/10.1021/jp307297s Shu, Y., Liu, M., Chen, S., Chen, X., Wang, J., 2011. New insight into molecular interactions of imidazolium ionic liquids with bovine serum albumin. J. Phys. Chem. B 115, 12306–12314. https://doi.org/10.1021/jp2071925 Sigma-Aldrich, 2018. Products information - Myoglobin [WWW Document]. URL https://www.sigmaaldrich.com/deepweb/assets/sigmaaldrich/product/documents /294/482/m0630pis.pdf (accessed 3.9.22). Sigma-Aldrich, 2014. Product information - Insulin from bovine pancreas [WWW Document]. URL https://www.sigmaaldrich.com/deepweb/assets/sigmaaldrich/product/documents /132/262/i6634pis.pdf (accessed 2.21.22). Sigma-Aldrich, 1996. Product Information - Hemoglobin [WWW Document]. URL https://www.sigmaaldrich.com/deepweb/assets/sigmaaldrich/product/documents /400/152/h2500pis.pdf (accessed 2.21.22). Silva, M., Figueiredo, A.M., Cabrita, E.J., 2014. Epitope mapping of imidazolium cations in ionic liquid–protein interactions unveils the balance between hydrophobicity and electrostatics towards protein destabilisation. Phys. Chem. Chem. Phys. 16, 23394–23403. https://doi.org/10.1039/C4CP03534H 62 Journal Pre-proof Jo u rn al Pr e- pr oo f Singh, T., Bharmoria, P., Morikawa, M., Kimizuka, N., Kumar, A., 2012. Ionic liquids induced structural changes of bovine serum albumin in aqueous media: a detailed physicochemical and spectroscopic study. J. Phys. Chem. B 116, 11924–11935. https://doi.org/10.1021/jp303609h Sivapragasam, M., Jaganathan, J.R., Levêque, J.-M., Moniruzzaman, M., Mutalib, M.A., 2019. Microbial biocompatibility of phosphonium-and ammonium-based ionic liquids. J. Mol. Liq. 273, 107–115. https://doi.org/10.1016/j.molliq.2018.10.022 Sivapragasam, M., Moniruzzaman, M., Goto, M., 2016. Recent advances in exploiting ionic liquids for biomolecules: Solubility, stability and applications. Biotechnol. J. 11, 1000–1013. https://doi.org/10.1002/biot.201500603 Solanki, R., Rostamabadi, H., Patel, S., Jafari, S.M., 2021. Anticancer nano-delivery systems based on bovine serum albumin nanoparticles: A critical review. Int. J. Biol. Macromol. 193, 528–540. https://doi.org/10.1016/j.ijbiomac.2021.10.040 Stillwell, W., 2016. Membrane Biogenesis: Fatty Acids, in: An Introduction to Biological Membranes, Second Ed. Academic Press, Indiana, USA, pp. 315– 329. Summers, C.A., Flowers, R.A., 2000. Protein renaturation by the liquid organic salt ethylammonium nitrate. Protein Sci. 9, 2001–2008. https://doi.org/10.1110/ps.9.10.2001 Swann, M.J., Peel, L.L., Carrington, S., Freeman, N.J., 2004. Dual-polarization interferometry: an analytical technique to measure changes in protein structure in real time, to determine the stoichiometry of binding events, and to differentiate between specific and nonspecific interactions. Anal. Biochem. 329, 190–198. https://doi.org/10.1016/j.ab.2004.02.019 Takekiyo, T., Fukudome, K., Yamazaki, K., Abe, H., Yoshimura, Y., 2014a. Protein aggregation and partial globular state in aqueous 1-alkyl-3-methylimidazolium nitrate solutions. Chem. Phys. Lett. 602, 22–27. https://doi.org/10.1016/j.cplett.2014.03.089 Takekiyo, T., Koyama, Y., Yamazaki, K., Abe, H., Yoshimura, Y., 2013. Ionic liquidinduced formation of the α-helical structure of β-lactoglobulin. J. Phys. Chem. B 117, 10142–10148. https://doi.org/10.1021/jp405834n Takekiyo, T., Nihei, A., Yamazaki, K., Aono, M., Abe, H., Yoshimura, Y., 2014b. Optical spectroscopic studies on structural changes of helical-rich proteins in aqueous solutions of ionic liquids. J. Solution Chem. 43, 1701–1709. https://doi.org/10.1007/s10953-014-0226-8 Tamura, K., Nakamura, N., Ohno, H., 2012. Cytochrome C dissolved in 1‐allyl‐3‐ methylimidazolium chloride type ionic liquid undergoes a quasi‐reversible redox reaction up to 140° C. Biotechnol. Bioeng. 109, 729–735. https://doi.org/10.1002/bit.24357 Tan, Z., Liu, J., Pang, L., 2012. Advances in analytical chemistry using the unique properties of ionic liquids. Trends Anal. Chem. 39, 218–227. https://doi.org/10.1016/j.trac.2012.06.005 Todinova, S., Guncheva, M., Yancheva, D., 2016. Thermal and conformational stability of insulin in the presence of imidazolium-based ionic liquids. J. Therm. Anal. Calorm. 123, 2591–2598. https://doi.org/10.1007/s10973-016-5287-z Tsien, R.Y., 1998. The Green Fluorescent Protein. Ann. Rev. Biochem. 67, 509–544. https://doi.org/10.1146/annurev.biochem.67.1.509 Uddin, S., Chowdhury, M.R., Wakabayashi, R., Kamiya, N., Moniruzzaman, M., Goto, M., 2020. Lipid based biocompatible ionic liquids: Synthesis, characterization 63 Journal Pre-proof Jo u rn al Pr e- pr oo f and biocompatibility evaluation. Chem. Comm. 56, 13756–13759. https://doi.org/10.1039/D0CC04491A UN – United Nations, 2022. UN Sustainable Development Goals [WWW Document]. URL https://sustainabledevelopment.un.org/?menu=1300 (accessed 3.8.20). UniProt, 2020. UniParc [WWW Document]. URL https://www.uniprot.org/uniparc/ (accessed 9.22.20). Vasantha, T., Kumar, A., Attri, P., Venkatesu, P., Rama Devi, R.S., 2014. The solubility and stability of amino acids in biocompatible ionic liquids. Protein Peptide Lett. 21, 15–24. https://doi.org/10.2174/09298665113209990071 Vashishat, R., Chabba, S., Mahajan, R.K., 2017. Surface active ionic liquid induced conformational transition in aqueous medium of hemoglobin. RSC Adv. 7, 13041–13052. https://doi.org/10.1039/C7RA00075H Ventura, S.P., e Silva, F.A., Gonçalves, A.M., Pereira, J.L., Gonçalves, F., Coutinho, J.A., 2014. Ecotoxicity analysis of cholinium-based ionic liquids to Vibrio fischeri marine bacteria. Ecotox. Environ. Safe. 102, 48–54. https://doi.org/10.1016/j.ecoenv.2014.01.003 Veríssimo, N.V., Nakamura, C.N., de Oliveira, F., Kuhn, B.L., Frizzo, C.P., Pereira, J.F., Santos-Ebinuma, V.C., 2022. Effect of amphiphilic ionic liquids on the colorimetric properties of polyketides colorants. J. Mol. Liq. 119857. https://doi.org/10.1016/j.molliq.2022.119857 Veríssimo, N.V., Saponi, C.F., Ryan, T.M., Greaves, T.L., Pereira, J.F., 2021. Imidazolium-based ionic liquids as additives to preserve the Enhanced Green Fluorescent Protein fluorescent activity. Green. Chem. Eng. 2, 412–422. https://doi.org/10.1016/j.gce.2021.08.001 Vieira, N.S.M., Castro, P.J., Marques, D.F., Araújo, J.M., Pereiro, A.B., 2020. Tailormade fluorinated ionic liquids for protein delivery. Nanomaterials 10, 1594. https://doi.org/10.3390/nano10081594 Wakayama, R., Uchiyama, S., Hall, D., 2019. Ionic liquids and protein folding—Old tricks for new solvents. Biophys. Rev. 11, 209–225. https://doi.org/10.1007/s12551-019-00509-2 Walsh, G., 2013. Biopharmaceuticals: Biochemistry and biotechnology, 2nd ed. John Wiley & Sons, Chichester, England. Wang, D., Zhao, X., Shen, T.-J., Ho, C., Spiro, T.G., 1999. Role of interhelical H-bonds (Wα14− Tα67 and Wβ15− Sβ72) in the hemoglobin allosteric reaction path evaluated by UV resonance raman spectroscopy of site-mutants. J. Am. Chem. Soc. 121, 11197–11203. https://doi.org/10.1021/ja992228w Wang, J., Zhang, B., 2018. Bovine serum albumin as a versatile platform for cancer imaging and therapy. Curr. Med. Chem. 25, 2938–2953. https://doi.org/10.2174/0929867324666170314143335 Wang, S.-F., Chen, T., Zhang, Z.-L., Pang, D.-W., Wong, K.-Y., 2007. Effects of hydrophilic room-temperature ionic liquid 1-butyl-3-methylimidazolium tetrafluoroborate on direct electrochemistry and bioelectrocatalysis of heme proteins entrapped in agarose hydrogel films. Electrochem. Comm. 9, 1709– 1714. https://doi.org/10.1016/j.elecom.2007.03.018 Wang, S.-F., Chen, T., Zhang, Z.-L., Shen, X.-C., Lu, Z.-X., Pang, D.-W., Wong, K.-Y., 2005. Direct electrochemistry and electrocatalysis of heme proteins entrapped in agarose hydrogel films in room-temperature ionic liquids. Langmuir 21, 9260– 9266. https://doi.org/10.1021/la050947k Wang, W., 2005. Protein aggregation and its inhibition in biopharmaceutics. Int. J. Pharm. 289, 1–30. https://doi.org/10.1016/j.ijpharm.2004.11.014 64 Journal Pre-proof Jo u rn al Pr e- pr oo f Wang, W., Nema, S., Teagarden, D., 2010. Protein aggregation—pathways and influencing factors. Int. J. Pharm. 390, 89–99. Wang, X., Liu, J., Sun, L., Yu, L., Jiao, J., Wang, R., 2012. Interaction of bovine serum albumin with ester-functionalized anionic surface-active ionic liquids in aqueous solution: a detailed physicochemical and conformational study. J. Phys. Chem. B 116, 12479–12488. https://doi.org/10.1021/jp307516a Ward, W.W., Prentice, H.J., Roth, A.F., Cody, C.W., Reeves, S.C., 1982. Spectral perturbations of the Aequorea green‐fluorescent protein. Photochem. Photobiol. 35, 803–808. https://doi.org/10.1111/j.1751-1097.1982.tb02651.x Watson, J.D., Laskowski, R.A., Thornton, J.M., 2005. Predicting protein function from sequence and structural data. Curr. Opin. Struc. Biol. 15, 275–284. https://doi.org/10.1016/j.sbi.2005.04.003 Wei, W., Danielson, N.D., 2011. Fluorescence and circular dichroism spectroscopy of cytochrome c in alkylammonium formate ionic liquids. Biomacromolecules 12, 290–297. https://doi.org/10.1021/bm1008052 Weingärtner, H., Cabrele, C., Herrmann, C., 2012. How ionic liquids can help to stabilize native proteins. Phys. Chem. Chem. Phys. 14, 415–426. https://doi.org/10.1039/C1CP21947B WHO - World Health Organization, 2020. Immunization [WWW Document]. URL https://www.who.int/news-room/facts-in-pictures/detail/immunization (accessed 8.25.20). Wittenberg, J.B., Wittenberg, B.A., 2003. Myoglobin function reassessed. J. Exp. Biol. 206, 2011–2020. https://doi.org/10.1242/jeb.00243 Wu, X., Zhu, Q., Chen, Z., Wu, W., Lu, Y., Qi, J., 2021. Ionic liquids as a useful tool for tailoring active pharmaceutical ingredients. J. Control. Release 338, 268– 283. https://doi.org/10.1016/j.jconrel.2021.08.032 Xiang, Z., 2006. Advances in homology protein structure modeling. Curr. Protein Peptide Sci. 7, 217–227. https://doi.org/10.2174/138920306777452312 Xu, J., Jiao, F., Yu, L., 2008. Protein structure prediction using threading, in: Protein Structure Prediction. Springer, New York, USA, pp. 91–121. Yan, H., Wu, J., Dai, G., Zhong, A., Chen, H., Yang, J., Han, D., 2012. Interaction mechanisms of ionic liquids [Cnmim] Br (n= 4, 6, 8, 10) with bovine serum albumin. J. Lumin. 132, 622–628. https://doi.org/10.1016/j.jlumin.2011.10.026 Yang, Z., 2009. Hofmeister effects: An explanation for the impact of ionic liquids on biocatalysis. J. Biotechnol. 144, 12–22. https://doi.org/10.1016/j.jbiotec.2009.04.011 Yang, Z., Yue, Y.-J., Huang, W.-C., Zhuang, X.-M., Chen, Z.-T., Xing, M., 2009. Importance of the ionic nature of ionic liquids in affecting enzyme performance. J. Biochem. 145, 355–364. https://doi.org/10.1093/jb/mvn173 Zaffran, M., Vandelaer, J., Kristensen, D., Melgaard, B., Yadav, P., Antwi-Agyei, K.O., Lasher, H., 2013. The imperative for stronger vaccine supply and logistics systems. Vaccine 31, B73–B80. https://doi.org/10.1016/j.vaccine.2012.11.036 Zhang, Y., Cremer, P.S., 2006. Interactions between macromolecules and ions: The Hofmeister series. Curr. Opin. Chem. Biol. 10, 658–663. https://doi.org/10.1016/j.cbpa.2006.09.020 Zhao, H., 2016. Protein stabilization and enzyme activation in ionic liquids: Specific ion effects. J. Chem. Technol. Biotechnol. 91, 25–50. https://doi.org/10.1002/jctb.4837 65 Journal Pre-proof Jo u rn al Pr e- pr oo f Zhao, Q., Chu, H., Zhao, B., Liang, Z., Zhang, L., Zhang, Y., 2018. Advances of ionic liquids-based methods for protein analysis. Trends Anal. Chem. 108, 239–246. https://doi.org/10.1016/j.trac.2018.09.008 Zhou, H., Ning, Z., E. Starr, A., Abu-Farha, M., Figeys, D., 2012. Advancements in topdown proteomics. Anal. Chem. 84, 720–734. https://doi.org/10.1021/ac202882y Zhu, L.-Y., Li, G.-Q., Zheng, F.-Y., 2011. Interaction of bovine serum albumin with two alkylimidazolium-based ionic liquids investigated by microcalorimetry and circular dichroism. J. Biophys. Chem. 2, 147. https://doi.org/10.4236/jbpc.2011.22018 Zimmer, M., 2002. Green Fluorescent Protein (GFP): Applications, structure and related photophysical behavior. Chem. Rev. 102, 759–782. https://doi.org/10.1021/cr010142r 66 Journal Pre-proof Declaration of interests ☒ The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. ☐The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Jo u rn al Pr e- pr oo f Declaration of interests: None 67 Journal Pre-proof Figure captions Fig. 1. A) Primary structure of proteins – amino acid chain. B) Secondary structure of proteins – interactions of polypeptide chains: α-helix, β-sheet, and random coil. C) The tertiary structure of proteins – three-dimensional folding of the protein structure (demonstrated by the structure of the wild-type Green Fluorescent Protein, PDB ID: 1GFL). D) Quaternary structure of proteins – packing of different subunits of protein (demonstrated by the Human hemoglobin A, PDB ID: 1MKO). Images of the proteins were produced with the PDB structures using UCSF Chimera 1.14 (Berman et al., 2002; Pettersen et al., 2004). oo f Fig. 2. Schematic representation of the main issues and potential solutions for expanding the large-scale access to proteins of commercial interest. Pr e- pr Fig. 3. Structure of A) Insulin (Human insulin, PDB ID: 3E7Y) and B) Hemoglobin (Human hemoglobin A, PDB ID: 1MKO). Images of the proteins were produced with the PDB structures using UCSF Chimera 1.14 (Berman et al., 2002; Pettersen et al., 2004). Jo u rn al Fig. 4. Structure of the hydrophilic proteins A) Cytochrome C from horse heart (PDB ID: 1HRC), B) Wild-type Green Fluorescent Protein (PDB ID: 1GFL), C) Human myoglobin mutant K45R (PDB ID: 3RGK), D) Bovine serum albumin (PDB ID: 4F5S), E) Human serum albumin (PDB ID: 1BM0), and F) Bovine β-Lactoglobulin A (PDB ID: 1CJ5). Images of the proteins were produced with the PDB structures using UCSF Chimera 1.14 (Berman et al., 2002; Pettersen et al., 2004). Fig. 5. Percentage of IL solutions that maintain or increase non-enzymatic protein stability according to Table 4. The effect of ILs on proteins was divided into A) Concentration, B) IL class, C) Ammonium ILs (concentration and protein type), and D) Imidazolium ILs (concentration and protein type). Fig. 6. Percentage of IL solutions that maintained or increased non-enzymatic proteins, according to Table 5, for the different types of protein stability parameters (structural, thermal, activity, and aggregation). For aggregation, improvement of stability includes ILs that decrease protein aggregation. Fig. 7. Percentage of IL solutions that maintained or increased the stability of the nonenzymatic proteins, according to Table 6, for each protein and concentration range (< 0.1 M, 0.0 – 1 M, > 1 M, and all concentrations). 68 Journal Pre-proof Fig. 8. Percentage of IL solutions that maintained or increased the stability of the nonenzymatic proteins, according to Table 6, for each protein and IL class (ammonium-, cholinium-, imidazolium-, pyridinium- and pyrrolidinium-based ILs, and all IL classes). Jo u rn al Pr e- pr oo f Fig. 9. SWOT (strengths, weaknesses, opportunities, and threats) analysis of the ILs application for protein stabilization. 69 Journal Pre-proof Highlights Compilation of the effects of ILs on the stability of non-enzymatic proteins. Tables were organized by protein properties, IL classes, and IL concentrations. Biocompatibility of ILs for biomedical applications. Perspective on the use of ILs as additives or solvents for proteins. Strengths, weaknesses, opportunities, and threats of ILs for protein stabilization. Jo u rn al Pr e- pr oo f 70 Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9
