Bioionic Liquids: Enabling a Paradigm Shift Toward Advanced and Smart Biomedical Applications

Ionic liquids (ILs) exhibit unique properties of good ionic conductivity, electrochemical and thermal stability, and nonflammability, which make them promising candidates for biomedical applications. The limitations of their cytocompatibility are enhanced by using bioionic liquids (BILs) derived from biological molecules such as amines, sugars, and organic acids. BILs can be synthesized using tailorable chemistries that enable their immobilization onto biopolymers. For example, the cholinium ion and its derivatives have found significant interest in tissue engineering and drug delivery systems. Ion‐doped BIL‐functionalized polymers and their composites can also be used to design pH and electrical responsive actuators and sensors. The cytocompatibility and low immunogenicity of BIL‐functionalized polymers enable the possibilities of their use for power storage devices as well as implantable devices. These devices are gaining recognition and importance in nucleic acid delivery and molecular medicine. This review focuses on the recent advances of BILs in biomedical applications. Specifically, the review explores BILs as agents for biopolymer functionalization and highlights BILs as solvents for supermolecular ionic networks.


Introduction
Ionic liquids (ILs) are organic salts with low melting temperatures (below 100°C), good ionic conductivity (within 10 À4 -10 À2 S cm À1 around room temperature), electrochemical and thermal stability, and nonflammability, making them promising materials for many applications. [1] Since the first report on the IL (ethylammonium nitrate) in 1914, ILs have been a major scientific area, with the pace of research accelerating especially within the last decade. Increased multidisciplinary studies have emerged, including chemistry, materials science and engineering, and chemical and environmental engineering. [1][2][3][4] A driving factor behind the plethora of applications stems from the various combinations of cations and anions that can be used to meet the scientific definition of ILs, which leads to a diverse range of physicochemical behaviors. [1,2] Their relatively low vapor pressures compared to conventional organic solvents make them desirable for green processes where scrubbing of solvent vapors needs elimination as a unit operation. [2] ILs have been further delineated into applications that utilize them as functional pendant groups in polymeric structures, or in supported onto membranes and metal-organic frameworks. Their hybrid ionic-organic nature makes them suitable for certain catalytic activities or as cocatalytic support with potential applications in supercapacitors, ion gels, separation, or lubrication. [1,2] ILs have been particularly used for catalytic conversion of cellulosic and lignocellulosic biomass and their valorization into biofuels and biorenewable chemicals. [2,3] Thermoresponsive ILs have been used for catalyzing hydroformylation reactions proffering a dual Ionic liquids (ILs) exhibit unique properties of good ionic conductivity, electrochemical and thermal stability, and nonflammability, which make them promising candidates for biomedical applications. The limitations of their cytocompatibility are enhanced by using bioionic liquids (BILs) derived from biological molecules such as amines, sugars, and organic acids. BILs can be synthesized using tailorable chemistries that enable their immobilization onto biopolymers. For example, the cholinium ion and its derivatives have found significant interest in tissue engineering and drug delivery systems. Ion-doped BIL-functionalized polymers and their composites can also be used to design pH and electrical responsive actuators and sensors. The cytocompatibility and low immunogenicity of BIL-functionalized polymers enable the possibilities of their use for power storage devices as well as implantable devices. These devices are gaining recognition and importance in nucleic acid delivery and molecular medicine. This review focuses on the recent advances of BILs in biomedical applications. Specifically, the review explores BILs as agents for biopolymer functionalization and highlights BILs as solvents for supermolecular ionic networks.
action as a solvent and catalyst. [2,3] ILs have been employed in separation processes with high separation efficiency, particularly extraction and purification of bioactive compounds and molecules with complex solubility profiles. [2,3] The ability for ILs to be supported as excellent solvents or impregnated liquid membranes or as chemical functionalities renders them useful in liquid-liquid extraction as well as solid-liquid extraction, solid-phase extraction, and induced precipitation. [2][3][4] The wide electrochemical window and ionic conductivity of ILs have been used in electrochemistry and power storage applications. [4] Their interfacial and surface electrochemistry makes them suitable for usage in both composite electrode structures and high-capacitance electrolyte applications. [4] The poor biodegradability and environmental toxicity of ILs have limited their applications in wearable and implantable devices. Developmental research that focuses on biocompatible ILs has thus prompted the progress of novel compounds with enhanced toxicological and biodegradable profiles. [5,6] Such novel IL structures that employ moieties from amino acids, artificial sweeteners, glucose, and organic acids are termed as bioionic liquids (BILs). [7] Natural or synthetically derived biomacromolecules may form either the cationic or anionic moiety of the BILs. The first BIL, made by Fukumoto et al., was amino acid (AA)-derived IL, wherein the AA was coupled as an anion, either with imidazolium cation or with an appropriate cationic counterpart. [8] Imidazolium cation-based ILs have been derived from D-fructose as well as polysaccharides. [9,10] Similarly, lignin or hemicellulose structures have also been converted or derived into cationic or anionic counterparts of ILs. [11] Carboxymethylated chitosan polymers have been used as polyanionic counterions to imidazolium cations. [12] The presence of either cation or anion that maintains the structure of the biomolecular entity from which it is derived ensures a metabolic pathway for biodegradation. This biodegradability endows them with a high degree of biocompatibility, which can be harnessed for further biological applications.
The water-soluble cholinium cation is common in biodegradable, cytocompatible, and cost-effective BIL structures. [13] Choline is a precursor of cellular phospholipids, such as phosphatidylcholine and sphingomyelin. [14] Choline degrades to small nontoxic chain molecules, which has enhanced its appealing potential for biological applications. For example, cholinium cation-based BILs have been investigated with various counteranion combinations, including active pharmaceutical ingredient derivatives such as ampicillin, salicylic acid derivates, and picolinic acid derivates. These currently established systems showed a significant improvement in the capacity of cell membrane penetration with concomitant drug delivery possibilities. Glycine betaine-based BILs are alternatives to cholinium-based BILs, owing to their abundance in sugar molasses and similarities in structure with cholinium via trimethyl alkylammonium and carboxylate structures. [15] However, their applications are limited to acting as solvents in green synthesis to date. In this review, we focus on the use of BILs in biomedical applications, entailing tissue engineering, bioprinting, and bioelectronics. The review also explores BIL-based supermolecular chemistries that have been utilized in the biomedical field. The review is based on searches of journal articles, books, and patents over, but not necessarily limited to, the years 2001-2022, primarily searched using Google Scholar, science.gov, PubMed, and ScienceDirect. The review has concentrated on biomedical applications with a particular focus on BILs in biomedical applications.
A schematic of the application fields is shown in Figure 1a,b. [16] A large amount of current research and developmental work using BILs remains an indispensable asset for various biotechnological  [15] Copyright 2019, Royal Society of Chemistry. b) Biomedical applications of ILs. [14] c) BIL structures most often used as active pharmaceutical ingredients-cholinium cation is often paired with anions derived from the structures shown. [18] transformations, followed by their applications in electrochemistry, chemical catalysis, and pharmaceutic applications. To a limited extent, BILs have been used in tissue engineering as solubilizers and stabilizers in drug delivery systems because of their improved bioavailability. The functionalization of BILs into hydrogels has expedited a variety of applications, such as pH and electrical stimuli-responsive actuators and sensors and multiresponsive systems for programmed drug delivery platforms. In addition, BILs are incorporated into natural and synthetic polymer matrices as supported liquids or in the form of functionalization of the polymer backbone. Such structures have been used in biomedical devices or power storage applications. [17,18] The tailorability of BIL-functionalized polymers has led to complex engineered structures, including artificial muscles or low immunogenic tissue engineering scaffolds. [7,13] 2. Cationic, Anionic, and Zwitterionic BILs ILs have been used as green solvents owing to their nonflammability and low vapor pressure. [19,20] The ionic counterparts can yield a wide variety of combinations, thus making ILs known as "designer" solvents. These structures exhibit high solvation, specificity, and selectivity for separation processes and are advantageous in drug delivery, plasticization, catalysis, and polymer processing. Their wide electrochemical potential window (which can be as high as 5.7 V between platinum electrodes) makes them particularly excellent candidates for electrochemical and photovoltaic applications. Biocompatible anions or cations used in BILs lead to metabolic degradation products of relatively lower toxicities. For example, the cholinium cation can be metabolized in the body through several pathways and absorbed for essential functions. This makes the use of choline in BIL structures a safe proposition for in vivo biomedical applications without incurring toxic or complex metabolic by-products of degradation. [19,20] In 1998, choline was added as a B vitamin to human vitamins. [21] It is a precursor to the neurotransmitter acetylcholine and participates in several biological functions entailing muscle control and memory. The cholinium cation, often used as a part of BIL structures, is also a segment of cellular phospholipids phosphatidylcholine, and sphingomyelin, which is often present in bilayer cellular membranes. [22,23] Since the early 2000s, there has been a significant increase in the research work on cholinium cation-based BILs. The tunability and bioactivity of cholinium-based BILs have enabled their use as active pharmaceutical ingredients (APIs). [18,24] In such systems, the cholinium cation is accompanied by an anion derived from the API structures such as nalidixic acid (NAL), phenytoin, ampicillin, niflumic acid (NIF), pyrazines acid (PYR), 4-aminosalicylic acid (PAS), and picolinic acid (PIC). [25] The structures often used as APIs are shown in Figure 1c. APIs thus are conjugated with cholinium cations, forming a BIL structure, displaying better solubility and cellular penetration, and therefore resulting in novel and improved therapeutic platforms. [18,24] Good's buffers (GBs) have also been explored as counteranions to BILs. [19] GBs are typical zwitterionic biological hydrogen ion buffers, between pH 6 and 8, with high buffering capacities and low toxicities. [24] Matias and co-workers reported novel cholinesulfonate BIL buffers with the potential to dissolve cytochrome c (Cyt c) in its reduced state, acting as a stabilizer and preventing the protein from unfolding during isolation. [26] BILs have been used to enhance the electrochemical response of materials with low electrochemical conductivities by either physically mixing or chemically functionalizing the base material with BILs. [27] For example, cholinium-functionalized chitosan hydrogels have been used in multiresponsive drug delivery systems due to their superior electrical and pH sensitivities. [28] In a study reported in 2014, a choline nitrate-chitosan polymer gel electrolyte was used in a thin-film battery with robust mechanicals and excellent ionic conductivity appropriate for possible implantable applications. [29] BILs have been used in ionic gels for wearable bioelectronics, such as epidermal sensors, biosutures, and neuromechanical interfaces. [27] Their hydrogenbonding ability makes BILs excellent plasticizers for biopolymers such as starch. [3,4] Figure 2 summarizes commonly used cations, anions, and alkyl chain substituents employed in BIL structures. [30] 3. BILs as a Strategy for Enhancing Electrical

Conductivity of Biopolymers
The electronic and ionic conductivity of hydrogels and biopolymers have been used widely in biomedical applications. Despite their low impedance, conductive polymers suffer from poor mechanical performance, low cohesion, and delamination. [31] The conductivity of biopolymers and hydrogels has been commonly improved by incorporating conductive materials such as gold or silver nanoparticles (AuNPs or AgNPs), graphene, carbon nanotubes (CNTs), and conductive polymers (i.e., polyaniline, polypyrrole [PPy], and thiophene polymers) into hydrogel networks. [32][33][34][35] Despite their biodegradability, these composite hydrogels have poor biocompatibility. Hydrogel-AuNP-poly (N-isopropyl acrylamide) (PNIPAAm) nanocomposites were reported to exhibit thermoswitchable electrical properties. The AuNPs were functionalized with a vinyl group that would help them covalently bond to the polymer chains. The presence of these AuNPs improved the electrical conductivity of BILs by two orders of magnitude. [36] In another study, Dai et al. fabricated stimuli-responsive and self-healing AgNPs hydrogels. The rapid response was predicated on pH and temperature sol-gel switching. Similar to the previous study, the incorporation of AgNPs into polymeric networks has been seen to influence the mechanical, swelling, and thermal response of electroconductive hydrogels (ECHs). [37] Despite these advantages, cytotoxicity makes their in vivo applications uncertain.
Another method to improve the electrical properties of ECHs involves adding CNT dispersion to polymer solutions. Xu et al. developed a multifunctional ECH made of polyvinyl alcoholborax cross-linked hydrogel networks and cellulose nanofibercarbon nanotube (CNF-CNT). The approach brought together the conductivity of CNTs and the templating ability of CNFs. The resulting assembled solid-state supercapacitor showed a 117.1 F g À1 specific capacitance and a 96.4% retention of capacitance even after 1000 cycles. [34] Despite electromechanical advantages, the hydrophobicity of CNTs makes it challenging to incorporate them into ECHs. Zheng et al. used hyaluronic acid hydrogel and single-wall CNT composites for microactuator structures. [38,39] Intrinsically conducting polymers such as blends of poly(3,4-ethylene dioxythiophene) and poly(styrene sulfonate) have also been studied for sensing and stimulation. The blends showed electrical conductivity of 20 and 40 S cm À1 in PBS and DI water, respectively. Although poor biocompatibility was the main disadvantage, the hydrogel composites showed high strain, flexibility, and stability in wet physiological environments. [40] BILs have been used to minimize the toxicity induced by conductive nanoparticles or polymers. The biocompatibility of BILs is incumbent on the structure of the counterions. Ammonium, pyridinium, and imidazolium-based cation structures, such as propylimidazolium (PMIM) and propylmethylpiperidinium (PMPIP), have been paired with various anions and used as dispersants in polyvinylidene fluoride (PVDF) matrices to enable usage in sensors and actuators. [41] Electrical conductivity generally decreased with the increased length of the cation alkyl chains. The bending response in these actuators was attributed to ionic diffusion inside the polymer hydrogel. Ribeiro et al. reported improved electrical conductivity of the ionic electroactive materials using similar PVDF-imidazolium and trimethylammonium IL composites, which can be used as electromechanical sensors. [41] Similarly, levan polysaccharides have been plasticized using cholinium malate BILs, concomitant with ester bond formation between levan polysaccharide and [Ch][MA] BIL under bending. [42,43] This strategy is summarized in Figure 3.
Other similar strategies have been employed with choliniumbased BILs and deep eutectic solvent (DES) structures, where they were used as plasticizers in polymeric matrices, in effect, yielding enhanced electrochemical properties in such composites. Cholium-based BILs with low toxicity counteranions were used to plasticize starch thin films, which had a significant effect on the conductive, thermomechanical, and recrystallization behavior of the films. [44] In another study, potato starch-based films were plasticized using a cholinium-based BIL-glycerol or Figure 2. a) Commonly used cations, anions, and alkyl chain substituents for BILs. Reproduced with permission. [30] Copyright 2020, Elsevier. b) Synthetic pathways for making BILs from various renewable/biological sources. Reproduced with permission. [15] Copyright 2019, Royal Society of Chemistry.
urea mix as a DES with similar results. [45] As an alternative strategy, choline dihydrogen phosphate-based BIL and 1-butyl-3methylimidazolium dihydrogen phosphate-based ILs were synthesized in the form of a novel class of proton-conducting ionic plastic crystals. The plastic crystalline phase showed much higher ionic conductivities than either BIL or IL alone, promising possible applications in proton-conducting fuel cells that are required to have fast proton transport in the solid state. [46] A similar strategy was used in plastic crystals made from choline dihydrogen phosphate-based BILs with phosphoric acid. These plastic crystals had a higher proton conductivity compared to the pure BILs, which was confirmed with impedance spectroscopy. [47] For in vivo applications, ECHs are employed as a possible strategy, and they are fabricated by incorporating conductive materials into a tunable polymer backbone. They can be fabricated as per structural applications while utilizing both electronic and ionic conductivity. [31] These ECHs can be employed in electrostimulated drug delivery systems, neural and muscle cell cultures, bioconductors, sensors and actuators, prostheses, and artificial muscles. [27,29,31] For example, Noshadi et al. engineered ECHs by functionalizing nonconductive polymers with cholinium cation-based BILs, which could then be photopolymerized to obtain a tailorable hydrogel platform. The ECH demonstrated electrical and biocompatible properties akin to native cardiac tissue for 2D and 3D cardiac cell cultures. The incorporation of BILs onto the hydrogel platform also enhanced cardiomyocyte beating frequency. Annabi et al. electrospun a BIL-modified gelatin hydrogel platform for physio-electrochemical properties akin to native myocardium without any significant changes to fiber properties upon the incorporation of BILs. The cardiopatches restored impulse propagation and promoted the growth and function of cocultures of cardiomyocytes (CMs) and cardiac fibroblasts (CFs) in an in vitro setup, with a superior contractile profile compared to gelatin methacryloyl (GelMA) scaffolds, leading to the development of patches for cardiac remodeling. [31] The formation of ECHs using cholinium-based BILs to functionalize biopolymers is shown in Figure 4a,b, while (c) shows the schematic of a solid-state electrolyte-organic transistor based on cholinium-based BILs. The strategy of using BILs as a part of the biopolymer or ECH structure with ensuing enhanced electrochemical as well as electromechanical properties has been delineated in the following sections and subsections exploring the biomedical applications of such materials in transistors, sensors, actuators, and power storage capacitors.

Transistors
The development of electronic devices using semiconductor materials needs efficient charge carrier control because such charge accumulation can cause chemical reactions or magnetic ordering. Additionally, the formation of electric double layers can cause issues like unwarranted discharge owing to the induced electric fields. Field-effect transistors (FETs) can employ IL-based gates that offer the advantages of a wide electrochemical window and superior physicochemical stability and transistor performance. [48] BILs, in particular, can be employed in the form of a supported liquid membrane or as a chemically integrated part of the biopolymer solid-state electrolyte structure. For example, Kim et al. synthesized a levan polysaccharide and choline-based BIL used as electrolytes for biocompatible and biodegradable transistors using a solid-state electrolyte ( Figure 2a). The use of BIL imparted the fabricated transistors with the advantages of low volatility-due to the intrinsic low volatility of ILs, flexibility, and transparence in addition to better electromechanical properties with a specific capacitance of 40 μF cm À2 . Even at low driving voltage, it supported electrochemical ion transport with negligible hysteresis and mechanical reliability. [42] For example, electropotential signals such as the electrocardiogram (ECG) and electromyography (EMG) were effectively captured and measured from the human body with these transistors attached to human skin. They showed stable electrical performances even when bent or stretched with a superior signal-to-noise ratio. Therefore, BIL incorporation into polymeric networks is a plausible approach to obtaining electrical conductivity in hydrogel structures that can be used in biomedical applications. [42] 3.1.

Sensors and Actuators
An ideal sensor requires a broad working range, high accuracy, and sensitivity. The use of ILs in sensors was facilitated by the  www.advancedsciencenews.com www.advintellsyst.com development of poly (ionic liquids) (PILs), synthesized via the polymerization of monomeric IL and BIL units due to their good electrochemical and electromechanical properties. While ILs and BILs also proffer the additional advantage of low volatility hence making them safer to use in electronic applications, the use of PILs offers the processing advantages of a polymeric matrix without the experience of the IL/BIL leaching out of the matrix and sensor device. PILs also employed AuNPs and graphene NPs, photopolymerized in imidazolium PIL matrices, thereby creating structural tunnels as conductive pathways. These have been studied in glucose sensors with physisorbed glucose oxidase. [36,36,49] As another example, graphene composites with imidazolium-modified PIL or amine-terminated PIL, based on amino acids, were employed as a highly sensitive glucose detector. [29,50] Similarly, graphene oxide (GO)-PPy-based PILs have been used for dopamine sensing, where they functioned as an aid for nanosheet dispersion. [35] ILs, such as 1-butyl-3-methylimidazolium chloride ([BMIm][Cl]), dispersed in biomaterials (i.e., silk) for use in electrochemical biosensors were proven to support cellular proliferation while also being studied successfully as biocatalysts in Figure 4. Electrochemical hydrogels formed from immobilizing BIL onto a biopolymer backbone using specific chemistry. The ECHs can have tunable properties depending upon the biopolymer and extent of BIL functionalization and can be fabricated as per application requirements: a) acrylation of choline bicarbonate to form BIL-based choline acrylate (I). The panel also shows the conductivity of the polymers functionalized with this hydrogel in a two-probe electrical station setup (II, III). A schematic of ex vivo experiments performed using rat abdominal muscle tissues, using the BIL functionalized biopolymeric hydrogels, is shown (IV). The panel also shows the threshold voltages at which contraction was achieved (V, VI, VII). Reproduced with permission [25] Copyright 2014, Royal Society of Chemistry. b) A new platform of photocurable ECHs made by functionalizing nonconductive polymers with conductive cholinium acrylate-based BILs. Reproduced with permission. [135] Copyright 2020, Elsevier. c) Schematic of a solid-state electrolyteorganic transistor (choline-based IL as electrolyte). Reproduced with permission. [91] Copyright 2019, American Chemical Society.
www.advancedsciencenews.com www.advintellsyst.com amperometric or voltammetric biosensing of enzymes. [51][52][53] For example, heme proteins entrapped in 1-butyl-3-methylimidazolium hexafluorophosphate ([BMIm][PF6])-agarose hydrogel films were used for hemoglobin (Hb), Cyt c, myoglobin, and HRP detection. [54] Composite pastes of multiwall carbon nanotubes (MWCNTs) or carbon paste electrodes (CPEs) with various imidazolium-based ILs were dispersed in biopolymer hydrogels that are used for the detection of dopamine, where rapid electron transport occurs at electrodes without significant interference from ascorbic and uric acids. [55][56][57][58] In order to circumnavigate the cytotoxicity of imidazolium ILs, some works have used phenylalanine amino acid-based BILs for electrochemical oxidation in glucose sensing, with the intent of being used in implanted devices. [27] Biopolymer-choline BILs dispersions have been used for in vivo detection of lactic acid. The BILs were seen to have a direct effect on the protein unfolding and aggregation dynamics owing to the ion effects of ILs on the biocatalytic activity of the enzyme. [59] In addition to the advantages of low volatility, the wide electrochemical property window ensures a significantly large response to the electrical or chemical, or mechanical stimulus. This allows the BIL-biopolymer dispersion a wider experimental results window and hence superior accuracy. [59] When BILs are incorporated in polymeric structures, the additional advantage of tailorability of the structure can be correlated to the response, and the structure may be functionalized according to the required electrochemical or electromechanical response to input. While conductive hydrogels have been used for flexible electronics and devices, some may be hesitant to use them in vivo because they do not possess any antimicrobial or antifouling properties, which makes their fouling of imminent concern. Choline amino acid-based PILs have been developed to provide the dual advantages of metal coordination and electrostatic interactions and thus exhibited excellent self-healing ability, high fatigue resistance, and energy dissipation. The presence of the choline amino acid-based PILs endowed these polymers with antimicrobial activity and rapid strain sensing. The electrical response could remain stable even after 1000 cycles of stretching. These flexible devices have the ability to monitor a large number of subtle physiological motions in the human body, like writing (Figure 5a-c). Their sensory ability and biocompatibility make these PILs promising in sensors and robotics. [60] Cytocompatible PILs based on cholinium derivatives have been used to exfoliate graphene and form PILs/PPy/GO composite structures where the synergy between lamellar GO, conductive PPy, and cytocompatible PILs results in good electrochemical properties with stability, linearity, and high sensitivity. [35] The PILs are thought to change the surface charge properties toward electropositivity with subsequent improvement of aqueous dispersibility, electronic transmission, and electrocatalysis. Figure 5. Self-healing hydrogels and BIL-modified hydrogels as sensors and actuators. a) The self-healing process and mechanism of hydrogels and reversible dynamic bonds among BC (bacterial cellulose), PAAc (polyacrylic acid), and Fe 3þ , including hydrogen bonds and metal coordination. b) Relative resistance response of Cho-Trp0.8 hydrogel sensor under different pressure and schematic illustration of Cho-Trp0.8 hydrogel assembled into a flexible touch keyboard. c) Demonstration of Cho-Trp0.8 hydrogel as a flexible sensor with strain-responsive conductivity. Relative resistance variation of Cho-Trp0.8 hydrogel sensor in response to large-scale human motions, including finger, wrist, elbow, and knee. Reproduced with permission. [60] Copyright 2022, Elsevier.
www.advancedsciencenews.com www.advintellsyst.com For biomimetic and biocompatible actuator applications, the material must display softness while being electroactive. Though conventional ILs have been used with soft biopolymers, their cytotoxicity prevents them from their further use for long-term implantable applications. In 2020, Elhi et al. prepared PPy-PVDF using various choline-based BILs, with low environmental and cytotoxic impact. The actuators were demonstrated to be viable candidates for soft robotics. Choline acetate and choline isobutyrate-based BILs, among others, showed the highest strain difference and outperformed imidazolium IL-based systems. The toxicity of the BILs and the polymer composites were tested on bacterial strains and HeLa cells. The BIL cytotoxicity was low, though incumbent on the counteranion. Thus, these prove to be promising candidates for fabricating biocompatible soft polymer actuators. [61] In a similar study, gelatin was used as a base by Elhi et al. for building blocks of soft electroactive polymer actuators, which have the potential for application in biomimetic applications. In this study, PPy-gelatin blends were used along with choline-based BILs and compared to PVDF-imidazolium IL blends. Molecular dynamics studies highlighted cation clustering as a good predictor of the strain difference of the actuator. The group also fabricated a trilayer actuator using disk diffusion. The gelatin choline-based BIL was more elastic and more biocompatible. [62]

Power Source and Energy Storage
As demand for wearable and implantable devices has risen, the research in internal energy storage systems has followed suit to both miniaturize and make them more biocompatible. BILs offer the advantage of higher capacitance that allows for rapid charging, although their energy storage capacity still remains lower than that of traditional batteries. [63] In a research work reported in 2018, GO and reduced GO (rGO) were annealed using choline chloride-based BIL and urea to make a supercapacitor with a capacitance of 383 Fg À1 . A comparative structure without the use of the BIL showed capacitance of 321 and 196 Fg À1 , for nitrogen-doped reduced GO and undoped reduced graphene oxide, respectively, demonstrating the possibility of application of BILs in energy storage. [63] In a similar study, an environmentally friendly supercapacitor was fabricated using aluminumcoated paper as a substrate and activated carbon electrodes with choline chloride-based BIL and choline chloride-based DES. These supercapacitors had a larger operating window and were cost-effective and printable. Their charge-discharge characteristics were comparable to imidazolium-based IL electrolytes. Choline chloride with ethylene glycol showed the highest capacitance and power densities, performing better than imidazolium IL-based structures. [64] Choline nitrate-based BIL was used in conjunction with silk fibroin as an electrolyte in a biodegradable thin-film magnesium battery. The corresponding specific capacitance was 0.06 mAh cm À2 and the entire device was enzymatically degradable. The BIL-based battery system is a promising tool for next-generation biodegradable power sources, especially for transient medical bionics. [65] In situ 3D printability of implantable bioelectronics needs an integrated power source that can be bioprinted too. The power storage system should be based on an electrolyte that does not leak in vivo, as this would result in a loss of activity. In 2021, Krishnadoss et al. developed a novel electrolyte based on choline-based BIL functionalized hydrogel polymers. As the cholinium BIL is chemically bound to the biopolymer, it cannot diffuse out of the biopolymer. [66] These polymers were successfully printed into interdigitated biocompatible soft microsupercapacitors along with graphene hydrogel electrodes. Figure 6a-c shows the electrolyte synthesis, electrical properties, photographs of printed microsupercapacitors, and a comparison of its energy and power densities to standards. The electrolyte had a specific capacitance of %200 Fg À1 , while the supercapacitor had a specific capacitance of %16 μ Fg À1 and cyclic stability up to 10 000 cycles. The energy densities were nearly as high as implantable batteries, with the power density level of implantable supercapacitors, making these BIL functionalized structures promising for in situ 3D printed flexible bioelectronics with an integrated power source. [66] Another method to mitigate the hazards of leaching BILs from polymer matrices is through an ion gel structure. Researchers made gelatin-ethyl-3-methylimidazolium acetate-based cross-linked ion gels for the purpose of developing stretchable and self-healable energy storage devices for flexible and implantable electronics. [67] These electronics showed excellent self-healable and mechanical properties, ionic conductivity, and temperature-dependent specific capacitance. While polymer matrices and solvents often cause flammability and explosion hazards, the issue can be mitigated using BIL-polymer ion gels. These ion gels can be cross-linked or bonded into the biopolymer to prevent poor mechanical stability and leaching. [67] Safety concerns have prompted cholinium or ammonium BIL-based polymer electrolytes' development with soft solid features incorporated in a lithium-ion battery. A good example of this would be 1-ethyl-3-methylimidazolium dicyanamide with PVDF-HFP-based thin film laminated polymer-BIL electrolyte structures, where the BIL is either dispersed in the polymer or functionalized onto the polymer structure. [68] In this case, the conductivity is incumbent on the liquid phase. Oftentimes, the gel matrices used with BILs are made from polyethylene oxide (PEO)/polyacrylonitrile (PAN)/polymethyl methacrylate (PMMA). [69] Similar studies have also found that, by applying the appropriate BIL to gel matrices, the limitations imposed by the presence of reactive and volatile solvents can be mitigated.

Antimicrobial Properties of BILs and Their Biomedical Application
The rapid evolution of antibiotic resistance in microbes has given an impetus to the development of novel materials and strategies for its redressal. In order to meet the heightened clinical demand, local antibiotic applications need to balance drug delivery efficiency, cytotoxicity, and antibacterial efficacy. [67][68][69][70] Imidazolium-based PIL with antibacterial properties was grafted on titanium dioxide (TiO 2 ). This was photocured and ionexchanged with L-proline and L-tryptophan. The resulting structures had excellent activity against Staphylococcus aureus and Escherichia coli cytocompatibility and blood compatibility. [71] The common assumption is that a higher charge density of imidazolium cations and longer alkyl chain length endow remarkable antibacterial properties. Thermally sensitive antimicrobial hydrogels have been formed from the stereocomplexation of ABA block copolymers from L-lactide and ethylene glycol and a polycarbonate triblock functionalized with ammonium. [72] These can create micellar structures in water, with a substantial effect on the counteranion of the antimicrobial properties. [73] Assembled and aggregated structures based on BILs are discussed in greater detail in a later section.
Further development in this front is an antibiotic switch based on reversible supramolecular assembly between poly(p-phenylene vinylene) (PPV)-ammonium PIL and cucurbit, giving rise to a noncovalent complex with a hydrophilic exterior and hydrophobic interior cavity, which encapsulates quaternary ammonium ions, thus stabilizing the complex and reducing the biocidal activity of PPV. [73] Other strategies, like cationic liposomes exhibit antibacterial effects by preferentially adsorbing the negative cell wall via electrostatic interactions. [74] Polyvinyl alcohol (PVA)-tetrahydroxyborate (B(OH) 4 ) hydrogel with silver ions can act as a vector to make an antimicrobial multifunctional hydrogel, thereby expanding its biomedical applications. [75] It is thought that borate ester bonds between PVA and B(OH) 4 form a network junction, which endows the multifunctionality that spans the hydrogel's moisture retention to self-healing, syringe ability, and antibacterial effects ideal for wound healing.
Antimicrobial properties have often entailed incorporating metal nanoparticles as an approach of choice. Mandal et al. demonstrated that polyethylene glycol (PEG)/TritonX-100 capped AgNPs, incorporated into lyophilized collagen scaffold, showed gradual antibacterial activity on gram-positive and gram-negative Figure 6. Functionalization of Choline BIL to polymers and its simultaneous photocross-linking as a strategy to yield materials for high capacitance electrolytes: a) synthesis of choline BIL and its conjugation to methacrylated gelatin and PEG to yield BIL functionalized gelatin methacrylate (BG) and BIL-functionalized PEG diacrylate (BP). b) Galvanostatic charge/discharge curve of GH-L (working electrode) with PBS buffer, BIL-conjugated gelatin, and PEG (with 0% and 15% BIL loadings), as electrolyte at 0.1 A cm À 3 and À1 to 1 V, Tafel plot of the same hydrogels at 0-1 V, charge-discharge characteristics of uncross-linked BIL-functionalized gelatin and PEG as electrolytes with carbon electrodes. c) Bioprinted supercapacitor structures using the BIL-conjugated biopolymers, a sample of 3D-printed energy storage device connected to the 1 V power source to illuminate the LED, Ragone plot comparing energy and power densities of the microsupercapacitors made from the BIL-conjugated biopolymers. Reproduced with permission. [66] Copyright 2021, Elsevier.
www.advancedsciencenews.com www.advintellsyst.com bacteria. [68] Similarly, Chieu et al. studied AuNPs-cellulose/wool keratin (KER) composites and imidazolium-based ILs as support. [49] The biocompatible composite showed bactericidal capabilities with 97-98% growth reduction of antibiotic-resistant bacteria like Enterococcus faecalis and S. aureus. These were used for controlled drug delivery and to treat ulcerous infected wounds as dressings. Despite advantages, biologically significant physicochemical properties of NPs are challenging to manage and scale for practical applications. [49] Thus, ILs, particularly imidazolium-based ILs, were used as an alternative strategy. Jiang et al. synthesized quaternary ammonium salt of gelatin using 2,3-epoxypropyl trimethylammonium chloride (EPTAC). This epoxypropyl salt served as an antimicrobial polymer, containing thermoresponsive poly(N-isopropylacrylamide) (PNIPAM) and redox-responsive poly(ferrocenyl silane)-based hydrogels, which provide an excellent combination of antimicrobial resistance and biocompatibility for the targeted cells, offset only by their lack of stability. [69] Liang et al. studied imidazolium salts by loading them into hydrogels and testing for antimicrobial properties against S. aureus, Methicillin-resistant Staphylococcus aureus (MRSA), E. coli, and Pseudomonas aeruginosa PA01 bacteria, with up to 96.1% efficiency. [70] Huan et al. used antimicrobial imidazolium-based BIL-PVA cross-linked hydrogel with high strength, antibacterial, and antifungal abilities, which furthermore promoted cutaneous wound healing. Qin et al. developed pyrrolidinium-based polymeric ILs for antibacterial membranes with an efficiency that increased with an alkyl chain length of substitutions. [76] They demonstrated exceptional hemocompatibility and low cytotoxicity in vitro for use in topical applications.
While the use of imidazolium-based ILs is sufficient for antimicrobial applications in topical applications, they have an inherent cytotoxicity that prevents the ready proliferation of cells. This means that it cannot be readily used in corneal applications or in vivo implants, which need the proliferation of cells while allowing microbial resistance. Cholinium-, phosphonium-, and ammonium-based ILs have been studied for their antibacterial properties as possible alternatives for the ILs. Sivapragasam et al. studied microbial toxicity with phosphonium/ammonium cationic BILs where the anions were chosen from phenylalanine/ taurinate/hydroxide/ acetate. [77] The ILs exhibited EC50 values from 100.2 to 1000 mg L À1 and underscored that the anions were the primary contributors to the toxicity. In another study, cholinium-based ILs like choline phenylacetate demonstrated excellent inhibition of all microorganisms tested. [78] Salicylate, gallates, propionates, and peracetates of choline showed promising inhibitory performance on gram-positive and gram-negative bacteria and yeasts, in addition to being biodegradable, making them promising candidates for wound healing applications. [79][80][81] A similar work by Siopa et al. entailed choline BILs based on dimethylethanolamine monoquaternary ammonium salts and methyl diethanolamine, diethanolamine, and triethanolamine monoquaternary ammonium salts for studies on antibacterial resistance. It was revealed that the presence of a hydroxyethyl group along with a C14-C16 linker in a choline compound improved antimicrobial activity while also decreasing the BIL's cytotoxicity. [82] Choline and geranate (CAGE)-based BILs reported by Ibsen et al. identified the interaction mechanism with the gram-negative cell wall of E. coli. While CAGE has been used for topical infections, the use of choline-based BIL increased the antimicrobial potency, owing to choline's attraction toward the cell membrane, which allows for easy injection of CAGE into the microbe, thereby terminating the cells. [83] Similar research work in 2020 reported cholinium-based BILs with 5-dinitrosalicylate, gallate, 2-(4-isobutylphenyl)propionate, behenate, and peracetate anions. Throughout various studies, upon exposure to a variety of microorganisms, they were proven to have excellent antimicrobial resistance and cytocompatibility. [84] Cholinium-based BILs have been used in conjunction with zinc oxide (ZnO) nanoparticles in antibacterial formulations for topical applications. The application of the BIL to these nanoparticles both combats ZnO's tendency to agglomerate, and has a synergistic effect on ZnO's antibacterial properties. These differences make the usage of ZnO nanoparticles in conjunction with cholinium BILs promising alternatives to traditional antibiotic therapy. This same formulation was also shown to be biocompatible and nontoxic to normal keratinocyte cells, even under coculture conditions. [85] In another work, with choline-based BILs, aggregated micellar surfactant structures of choline laureate and choline oleate-based BILs were studied by Shah et al. for antimicrobial applications. The antimicrobial activity of the individual BIL, as well as that in a mixture, worked well against gram-positive and gram-negative bacteria. They also displayed low cytotoxicity in vitro with human C2C12 cell cultures. [86]

Hemostatic Properties of BILs and Their Biomedical Application in Tissue Adhesives and Antibacterial Dressings
The traditional tissue trauma repair and wound closure methods are beset by improper tissue integration due to poor biocompatibility. This imperfection often leads to poor wound healing and infections, an example of which is medical-grade cyanoacrylates. In addition to being biocompatible, biodegradable, or acting as a drug carrier, rigid biodegradable material adhesives need to be resilient and attach firmly to underlying tissue to prevent body fluid leakage and minimize invasion. A vital wound-healing adhesive property is that of hemostasis. Clotting, a vital process that provides the wound with a physical barrier, is incumbent on clotting agents, such as clotting factors, thrombin, and fibrinogen. Still, most of these hemostats are suitable for very shallow external wounds, and the need for hemostatic biomaterials, internal injuries, and deep cuts remains unmet.
Thrombin and fibrinogen-based injectable solutions pose the risk of activating coagulation in the circulatory system. To address this, researchers have studied polymer-based materials that can simultaneously exhibit both adhesive and hemostasis. Ryu et al. made hemostatic adhesives from injectable hydrogels using thermosensitive chitosan/Pluronic composites. The chitosan was functionalized using catechol groups, cross-linked with thiolated triblock Pluronic F-127. [87] Compared to the control, the total blood loss was brought down by nearly 75%, with these compositions, with easy setting under body temperature and good adhesion to soft tissue. Lih et al. studied rapid curing of chitosan-PEG hydrogels as hemostatic adhesives in similar www.advancedsciencenews.com www.advintellsyst.com work. [88] The hydrogels also showed improved skin incision healing relative to sutures, fibrin glue, or cyanoacrylate, suggesting promising applications for biomedical use. Other biopolymers like alkali polymerized, complexed, polydopamineÀsodium alginateÀpolyacrylamide (PDAÀSAÀPAM) hydrogels have been studied for skin tissue engineering. [89] They exhibited good adhesion to porcine skin with rapid blood coagulation, making them potential candidates for skin tissue engineering. Hong et al. studied adhesive hemostatic hydrogels for arterial repair and healing heart bleeds. These adhesive hydrogels were based on photoreactive gelatin methacrylate, N-(2-aminoethyl)-4-(4-(hydroxymethyl)-2-methoxy-5-nitrosophenoxy) butanamide, and glycosaminoglycan hyaluronic acid, which is a mimic for the extracellular matrix. The matrix was capable of withstanding pressures much higher than that of commercial fibrin glue. [90] The other class of material that has recently been studied in biomedical applications is the BIL-based bioadhesive structures. As a result of the abundance of positive charges in the structure, the anions' adhesive strength surpasses that of the cations' and is more capable of rapidly holding blood. Noshadi et al. reported novel hemostatic bioinspired multifunctional photocurable adhesives with choline-based BIL-conjugated biopolymers backbones, displaying excellent adhesion, biodegradability, and biocompatibility. The burst pressures increased by tenfold over the control and over 50% lowering of blood loss from a liver cut model over control sutures and staples, underscoring its apparent advantages. However, existing adhesives have limited functions and applications; BILs may contribute to building a new platform for the synthesis of novel multifunctional adhesives, augment the utility, and broaden their applications. Figure 7 illustrates the hemostatic properties of various IL/BIL-functionalized polymers and composites. Antimicrobial properties are especially important if the polymer serves as a tissue adhesive that allows for integration with the native tissue, where the polymer must have a structural similarity to the native tissue and must allow cellular proliferation in a watery body fluid-laden environment. The choline BIL structure is a precursor of a cellular phospholipid bilayer, leading to their excellent adhesion, thus allowing for cellular proliferation and integration with the tissue over time. At the same time, the polymer also demonstrated excellent antimicrobial resistance against gram-positive and gram-negative bacteria in both in vitro and in vivo infected corneal injury models, making it a promising approach for tissue repairs, wound dressings, and flexible electronic attachment to tissues. [91] In 2021, Das et al. studied the effect of various choline BILs─choline fumarate (Ch-Fu), choline adipate (Ch-Adi), choline caproate (Ch-Cap), choline caprylate (Ch-Capl), choline capriate (Ch-Capr), and choline laurate (Ch-Lau)─for selective coagulation of κ-carrageenan from Kappaphycus alvarezii seaweed extract obtained using water and alkali. Choline caprylate and laurate BILs displayed selectivity in coagulating the polysaccharide, promising implications for wound dressings. [92] Wound dressings need a critical optimization between bleeding control and wound healing. Often electrostatic interactions are utilized to drive dressing adhesion. In 2016, Yang et al. studied a choline phosphate BIL-functionalized cellulose membrane for arresting human red blood cells with potential application in wound dressing. The bioadhesion was driven by multivalent electrostatic interactions between phosphatidyl choline lipids heads on the cell membranes and their inverse but identical structures in the choline phosphate-based BIL that couples to the cellulose membrane. The cellulose membranes were functionalized onto the surface with polymer brushes containing choline phosphate BIL groups using surface-initiator atom transfer radical polymerization followed by click chemistry. Red cell binding was seen to be proportional to the density of choline-based BIL binding on the brushes. The red blood cells (RBCs) were seen to bind like a "bound pseudopodia"-membrane projections that distorted the RBC shape. This type of binding underscored the multivalent interactions between the RBCs and the choline phosphate BIL-functionalized cellulose membrane, with concomitant promising implications for hemostasis and trauma management (Figure 8a). [93] The mechanisms of hemostasis are thought to be multifarious. One of these mechanisms is the formation of a collagen network that traps the red blood cells. In 2020, Tarannum et al. studied the effects of choline BILs as biocompatible collagen cross-linkers and stabilizers. The hydration dynamics were probed using NMR relaxation and impedance measurements. Choline-based BILs cause a reorientation of the surrounding water milieu, casing compaction of collagen, pushing it toward cross-linking. The collagen scaffolds treated with BILs showed higher crosslinking and platelet attachment levels while retaining their cytocompatibility. Choline-based BILs thus acted like biocompatible crosslinkers of collagen with possible implications in biomedical applications (Figure 8b). [94]

Antifouling Properties of BILs and Their Biomedical Applications
Biomedical fouling is caused by the nonspecific adsorption of cells, proteins, and microorganisms onto the scaffold and implant surfaces, impairing their function as bandages for wound healing, biomedical devices, or biosensors. Generalpurpose antifouling strategies are challenging to be retrofitted for biomedical applications. Soft hydrogel-like surfaces have been designed to avoid excessive fouling by inhibiting adherence, which is a property that makes them susceptible to mechanical damage or exfoliation after long-term use or degradation from a host of environmental factors. [95][96][97] ILs are gradually gaining popularity as magnets in biofilm prevention. The mechanism by which their low cytotoxicity balances this antifouling activity is still an area of study. The advantage offered by ILs is their tunability of properties which can be modified to suit a given application environment. ILs range from imidazolium and quinolinium to cholinium cationic head structures that are being used as magnets. It has been seen that the modification of the cationic head is primarily responsible for driving the antifouling-cytocompatibility balance. Antifouling capability is one of the essential requisites for the targeted delivery of therapeutics or diagnostic particles. Thus, in addition to being used for enhanced cell penetration, cholinium BILs are being increasingly studied for their control of pathogenic biofilms. [98] Traditionally, robust solutions entailed composites like PVA and poly(2-acrylamido-2-methyl-1-propanesulfonic acid) (PAMPS)/polyacrylamide (PAAm) double network gels, which showed antifouling activity of hydroxy and sulfonic groups www.advancedsciencenews.com www.advintellsyst.com against barnacles. [97] Self-healing coatings made using disulfide functionalized hydrogels were reported for their antifouling activity. Some antifouling hydrogels were made from PEG methacrylates or N-hydroxyethyl acrylamide and 2-(methacryloyloxy)ethyl trimethylammonium chloride. [99] Researchers also developed mussel-inspired self-healing antifouling hydrogels, which were based on ABA triblock copolymers in which catechols and other aromatics provided them with rapid self-healing capabilities via hydrogen bonding. [97][98][99][100][101][102] The poor mechanical properties of hydrogels that limit their practical application were circumnavigated using BIL functionalization. Kostina et al. studied zwitterionic carboxy betaine methacrylamide and 2-hydroxyethyl methacrylate (HEMA)-Laponite dispersions as antifouling, self-healing, robust, and tough hydrogels. [100] Yang et al. studied salt-responsive zwitterionic antifouling polymer brushes of poly(3-(1-(4-vinyl benzyl)-1Himidazole-3-ium-3-yl)propane-1-sulfonate). [19] At appropriate ionic conditions, the brushes switched to yield surfaces with very little (<0.3 ng cm À2 ) protein adsorption and hence fouling and extremely low friction (u % 10 À3 ), thereby demonstrating their promising versatility in the medical field.
None of these traditional approaches satisfactorily achieved the antifouling cytocompatibility trade-off that biomedical applications entail. Thus, cholinium BILs were studied for the purposes of enhancing antifouling capabilities in liquid-infused porous surfaces. [13] Wylie et al. reported that slippery, super hydrophilic surfaces that had been infused with polyvinyl chloride (PVC) substrates were roughened by phosphonium Figure 7. Hemostatic properties of IL-polymers and composites-the application of various strategies to prompt hemostatic response in biopolymers a) PDA-catechol adhesive: adhesive strength of hydrogels to porcine skin; [87] b) tissue adhesive made by conjugating an acrylate derivative of choliniumbased BIL onto various biopolymer structures (gelatin methacrylate and PEG diacrylate): ex vivo and in vivo results, including puncture sealing and patching of a porcine heart wound. Measurement of burst pressure in the explanted heart. Blood loss volume estimated from in vivo tail cut model. BIL conjugation renders them highly efficient tissue adhesives with hemostatic properties. Reproduced with permission. [91] Copyright 2019, American Chemical Society. c) Schematic synthesis of PAA/Laponite NC gels. Reproduced with permission. [136] Copyright 2014, American Chemical Society.
www.advancedsciencenews.com www.advintellsyst.com and cholinium BILs, which could reduce bacterial adherence by up to four orders of magnitude within 24 h. [101,102] Targeted delivery of therapeutic and diagnostic particles is one of the most challenging applications to tackle, given the rapid in vivo fouling that prevents the particles from achieving their targeted efficacy or reaching their destination. Another issue is the rapid clearance of intravenously administered nanoparticles (NPs) from the in vivo system or bloodstream. Hamadani et al. used a special biocompatible protein-avoidant ionic liquids (PAILs) based on choline hexanoate BIL, as an NP surface modifier to stably coat poly(lactic-co-glycolic acid) (PLGA) NPs. The NPs showed a marked increase in resistance to in vitro protein adsorption and hence greater retention in the blood of mice. The choline hexanoate BIL also successfully redirected NP biodistribution to preferentially accumulate in the lungs, thus increasing possibilities of enhanced bioavailability with lower adjunct cytotoxicity, making it a promising candidate for targeted cancer drug delivery systems. [103] In another study, choline-based BIL monomeric units were radically polymerized into various biopolymer chains to deliver salicylates. These graft structures are assembled into spherical superstructures. The use of choline BIL significantly reduced the antifouling of these aggregates. The biodistribution was dependent on the chain length of the polymer. [104]

BILs and BIL-Functionalized Polymers in Bioprinting
Owing to trends toward the miniaturization of implantable and wearable biomedical devices, 3D bioprinting or additive manufacturing has been gaining popularity as a means of device fabrication, particularly in tissue engineering, flexible electronics, sensors, actuators, and power storage. PILs offer physical and viscoelastic properties along with the advantages of the intrinsic properties of polymer's processability, mechanical stability, and biocompatibility, thus making them popular in the bioprinting field as a versatile material.
PILs offer flexibility in molecular design for bioprinting that needs a delicate balance of processing and synergistic properties. PILs can be homopolymers, copolymers, or even cross-linked structures, including IPNs and SIPNs. Tethered and mobile charges allow for conductivity and charge stability, which are essential for device and energy storage applications. When utilizing 3D printing, the IL monomer can be polymerized with a catalyst, or a PIL structure can be cross-linked into an infinite network; for example, choline-functionalized methacrylate BILs could be in situ photocross-linked while being 3D printed. [42] Traditionally, when 3D printing phosphonium ionic monomers, the monomers are polymerized with diacrylate. [105] Click chemistry, which allows orthogonal combinatorial synthesis, has been used in copper-catalyzed azide-alkyne cycloaddition or thiol-ene and thiol-yne reactions for triazolium PILs. Sequential azide-alkyne coupling and Menshutkin reactions have been employed for a complex sequence of ions in PIL structures. [105] Imidazolium tetrafluoroborate PILs have also been 3D printed into piezoresistive tactile sensors. [106] PILs such as 1-ethyl-2-methylimidazolium tetrafluoroborate ([EMIM][BF4])/ 2-[[(butylamino)carbonyl]oxy]ethyl acrylate (BACOEA) can control composition and processing parameters, thus making it an appropriate choice for printable devices. [106][107][108] The ILs that Figure 8. a) Cellulose membrane functionalized with polymer brushes bearing multiple choline phosphate BIL groups-used as excellent hemostatic agents. Reproduced with permission. [92] Copyright 2016, Elsevier; b) choline-based BILs as a biocompatible cross-linker for collagen. Reproduced with permission. [93] Copyright 2020, Elsevier.
www.advancedsciencenews.com www.advintellsyst.com have been used ranged from biocompatible choline structures to imidazolium and a wide variety of combinatorial anions and cations, which offer flexibility to circumnavigate the biomedical challenges in drug delivery and the bioavailability of drug insolubility. BILs can be easily applied in inkjet 3D printing for biomedical applications such as printing of tissues and organs or even device printing of actuators and sensors (i.e., cellulose impregnated with phosphonium/cholinium or even [EMIM] acetate ([EMIM][OAc])). [109] The primary issue with the usage of other ILs is their cytocompatibility. Sometimes, this can be offset by using a biopolymer as the counterpolyanion with an imidazolium cation. However, it is the cation head that is primarily responsible for an IL's cytotoxicity. In order to diminish its cytotoxicity, a true BIL must be based on a biocompatible cation. For example, PVDF [EMIM] bis(trifluoromethylsulfonyl)imide [TFSI]) electrospun fibers have been used for tissue engineering, significantly affecting cell viability while maintaining their noncytotoxicity. [41] The same combination has been used in a polymer electrolyte-biocompatible magnesium-based implantable battery device. [110] The electrolyte also incorporated chitosan biopolymer, which enhanced its biocompatibility, ionic conductivity, and volumetric power density, pointing at a capability of supporting small implantable medical devices. Transparent and stretchable ionic conductors have been developed, based on poly(MMA-butyl acrylate) with IL [EMIM][TFSI] as an ionic skin-type strain sensor, to be used for human movement sensing, with remarkably high elongation limits and a recovery ratio of >96% even after 500 cycles. [111] Figure 9 illustrates IL and BIL-based formulations in sensors, power sources, and bioprinting.
ECHs have gained popularity in bioprinted biomedical applications as well. They are hydrated networks that can be modulated into highly tunable structures by the functionalization or incorporation of nanoparticles and conductive polymers. Biocompatible ECHs are often based on either blends or functionalized ILs with biocompatible polymers, where ILs can be grafted to the structure of hydrogels. Choline BIL grafted hydrogels combine the requisite properties of the BIL with the easy processability of the polymer for bioprinted applications such as tissue engineering, biosensors, flexible electronics, and other implantable medical devices. For example, Walker et al. synthesized a photocross-linked GelMA prepolymer, which was acrylated using a modified choline BIL structure. The hydrogels were accessible to bioprint and demonstrated tunable electrical conductivity within the range of native cardiac tissue. It was used to bioprint scaffolds with and without encapsulated cells, and maintained excellent viability of the cells encapsulated. The seeded cardiac cells displayed a higher beating frequency relative to the control GelMA hydrogel scaffolds, indicating that bioprinted BILs can be adapted to biomedical technology. [112][113][114][115] In a similar study, choline BILs were grafted onto PEG and gelatin structures to enhance the mechanical and electrical properties of the polymers. These were bioprinted into microsupercapacitors and studied as subcutaneous implants. The flow properties and ensuing device performance were seen to be directly correlated to BIL functionalization. [65,66] Figure 9 shows applications of various bioprinted conductive hydrogels and polymers functionalized with BILs.

The Applications of BILs in Tissue Engineering
In order to successfully engineer tissues, the materials used need to mimic biological materials. A common strategy entails culturing harvested autologous chondrocytes on collagen membrane and implanting them at the site of the injury. While insufficient vascularity acts as a hurdle to this approach, it can be improved by engineering microvasculature into the membrane scaffolds before implantation. By engineering these scaffolds to provide regenerative signals to cells, one can repair and regenerate tissues. This approach requires complex biomimetic biomaterials with multifunctional capability, in addition to bioinstructive, stimuliresponsive abilities. Biomaterials must transmit appropriate stimuli at tissue, cell, and subcellular levels with critical pathways, hence entailing cell adhesion complexes or contractile forces. This is especially true for muscle and neural cell development, which show significant differences when grown on rigid gels that mimic bone structure or brain tissue mechanicals. Heart cells require synchronous beating to grow, which paves the way for the requisite of a mechanosensitive scaffold. Additionally, the scaffold must permit biochemical signaling and deliver growth and angiogenic factors for vascularization, cytokines, and adhesion peptides for the neural supply. [41] Beyond these is the ability of a scaffold to keep itself from fouling and infection or, for that matter, be used as a system for delivering therapeutic genes. [41,90] Due to their porosity and high surface-to-volume ratio, nanofibers have been acknowledged as promising tissue engineering candidates. They are fabricated via electrospinning, which is commonly used for its simplicity and scalability. [41,90] Despite their difficulties in spinning, polyelectrolytes have been used to mimic natural extracellular matrix. The limitation of using them is their jet instability, which makes scaling a complex process. As most polyelectrolytes are water-soluble, electrospinning and process scaling become difficult due to the low volatility of water and its high surface tension as well as ion dissociation, leading to poor rheological control. [116] Often BILs or other salts are added to nonpolar polymers to alter their electrical properties, allowing their solutions to become conducting despite their insulating nature and hence facilitate their electrospinning. Herein, the electrospinning of biocompatible PIL could provide a superior recourse to materials that are adequately responsive to electrical stimulus while being spinnable, owing to their unique architecture. [30] Only a few PIL structures have been electrospun, even though PILs are organic soluble and suitable candidates for electrospinning. Poly(3-cyanomethyl-1-vinylimidazolium bis(trifluoromethanesulfonyl)imide) in blends with PVP, PVA, or PEO have been used to increase chain entanglements without viscosity alteration. [97] In electrospun fibers, the solvent also has a tremendous effect on the polymer's structure. In this context, electroactive electrospun PVDF fiber mat composites with 5-10% of and IL-based on 1-ethyl-3-methylimidazolium bis(trifluoromethanesulfonyl)amide were developed for biomedical applications. [41] Around 10% IL caused complete PVDF crystallization in the piezoelectric β-phase. This established that PVDF fiber properties could be tailored by differentiating the IL connect for integrated sensor and electromechanical actuator devices. Nevertheless, again, the limitation of cytocompatibility makes cellular proliferation and implantation an issue of concern.
www.advancedsciencenews.com www.advintellsyst.com BILs, as an alternative strategy, have also been used in muscle regeneration by employing active scaffolds with mechanoelectrical stimuli in a biomimetic microenvironment. In a study by Meira et al., imidazolium ILs and cholinium BILs acted as composites or blends with PVDF and were cast into thin films to fabricate ionic electroactive materials. The BILs employed were 2-hydroxyethyl-trimethylammonium dihydrogen phosphate [Ch] [DHP]. Researchers witnessed an increase in electrical conductivity and heightened PVDF crystallization in the polar β-phase. While the imidazolium ILs' performance overall surpassed that of the BILs', it was offset by the excellent cytocompatibility shown by the BILs with exceptional C2C12 cell proliferation. [117] Chitosan is a well-received cationic polyelectrolyte polymer that has been used in a wide variety of biomedical applications. In 2013, Brett et al. used chitosan, a cationic polyelectrolyte, with choline dihydrogen phosphate and choline chloride BILs to develop biocompatible and biodegradable materials for improved electrical and pH-sensitive properties. It was observed that a low pH (acidic) enhanced conductivity and the actuation capacity of the films, which was more pronounced with [Ch] [DHP]. These were studied as stimuli-responsive scaffolds for tissue engineering and other biomedical applications. The simultaneous effects of ILs on the electrical response of the films allowed for the development of biocompatible and biodegradable integrated iontophoretic systems for tissue engineering. [28] Polylactides (PLAs) are biopolyesters that possess promising biocompatible and biodegradable characteristics. However, their high hydrophobicity is of primary concern in tissue engineering applications. In a recent study, these limitations were circumvented by blending them with isotactic acid and choline taurinate. This blending caused partial aminolysis of the ester bonds of the polymer by the taurine amino group and led to the stabilized dispersion of oligomers. The grafted choline taurinate allowed for the modulation of pore dimensions in porous scaffold structures and the enhancement of hydrophilicity, thus making cellular proliferation attainable. [118] Choline BILs have been used earlier in collagenous matrices. They acted as cross-linkers for the collagen structure and exhibited excellent cytocompatibility with the possibility of being used for biomedical applications. [119] Choline acetate BIL and chitin have been used in the form of α-chitin-based 3D tissue engineering structures with porous morphology, interconnectivity, and excellent hydrophilic character. The cytotoxicity assays also underscored the biocompatibility of choline-based BIL when seeded with human adipose stem cells. [120] In a 2018 work, therapeutic DES, based on choline chloride BIL and ascorbic acid, was reported. Throughout a 6-month span of maintenance during which these BILs were applied to the solvents, researchers were able to stabilize dexamethasone and improve its cellular solubility while also sustaining cell viability. They proceeded to dope a starch blend with the dexamethasone solubilized eutectic solvent structure to yield a blend with encouraging traits that may be applied to the biomedical field in the future. [121] 9. Aggregated and Assembled BIL-Polymer Structures in Biomedical Applications Supramolecular self-assembly has the potential to produce responsive materials by tailoring building blocks and fabricating nanosystems. Organic building blocks are simple and easily modified and are highly responsive to dynamics encountered by self-assembled nanoarchitectures with concomitant enhanced sensitivity toward environmental stimuli─this, along with their simplicity and ease of modification, makes them particularly alluring in controllable drug release and bioimaging applications.
While they have been used as solvents and plasticizers, BILs and ILs have also been grafted onto the structures of a vast assortment of polymers to enhance biocompatibility. [28,122] These blends and grafted polymers often create aggregates and selfassemble formations that can be adapted to drug delivery systems. [122] For example, cellulose-poly(L-lactide) grafts synthesized in imidazolium-based ILs were self-assembled into micelles and used for sustained drug release. These assemblies had cores and shells as hydrophobic PLA and hydrophilic cellulose segments, respectively. [122,123] Similar imidazolium IL-polyacrylamidebased aggregated structures have been used for thermal and pH stimulus-responsive drug delivery. [124,125] ILs protonating groups like N,N-dimethylaminoethyl groups gave rise to the decontraction of the brush structure, reduced micellar diameter, and induced rupture of the micelle at a higher pH, thus making it a viable structure for pH-sensitive drug delivery. [126] Similar concepts have been used for drug uptake with chitosan-lilial conjugates made in imidazolium-based ILs, where the aggregate structure was seen to disintegrate at lower pH and could be used for lysosomal or endosomal uptake as a gradually acidified process. [127] ILs like (1-octyl-3-methylimidazolium acetate ([OMIM][Ac]) have been used for making biopolymer-particle suspension-based delivery systems that overcome the limitations of the size issue of NPs. [127,128] These structures were used in drug burst release studies with up to 80% sustained drug release for 10 h. Figure 10 illustrates some assembled and aggregated structures based on BIL-modified chitosan for biomedical applications.
In recent studies, cholinium and other ammonium ILs have been used as biocompatible alternatives to imidazolium ILs in aggregated structures. Drug nanocarriers composed of linear copolymers contained different contents of cholinium-based ILs like 2-[(methacryloyloxy)ethyl]trimethylammonium chloride. The chloride anions of the polymer chain were used for ion exchange with the pharmaceutical anions. These characteristics demonstrated that the polymer-drug ionic conjugates are suitable drug carriers with potential medical applications, particularly in treating lung and respiratory diseases. [129] Nevertheless, another approach to the formation of aggregates is the novel use of ester-functionalized surface-active ionic liquids (SAILs) based on nicotine BILs. These aggregates have been characterized for their micelle-forming capability as well as cytotoxicity toward C6-Glioma cells, making them potential future candidates for diverse biomedical applications. [130] Choline citrate BILs have been used for the transdermal delivery of hyaluronic acid. The skin is difficult to breach the barrier for drug delivery. In order to improve this process, BILs synthesized by neutralization reactions were used to form aggregates with hyaluronic acid and enhance transdermal delivery, which remained the same even with dilution. No irritation was noted in cutaneous mice studies, while the superficial moisture retention of the skin was enhanced, showing the potential use for facilitating the delivery of a wide variety of drugs, such as antiaging products to antivirals and antifungal drugs. [131] Figure 11 shows the application of cholinium BIL-based supermolecular aggregate strategies for nucleic acid and drug delivery applications.
Similar works have studied cationic vesicles for the encapsulation of curcumin. These vesicles were formed using imidazolium-based ILs and cholinium-based BILs through synergistic interaction between sodium butyrate. By increasing the hydrophobic characteristics of the vesicles, researchers discovered that it was directly related to the loading capacity and stability of the curcumin. The modification of the choline BIL cation could be used for enhanced potential sustained drug delivery or anticancer drug delivery. [132] A novel frontier of aggregated structure application is the encapsulation and delivery of nucleic acids. For example, DNA amphiphiles conjugated with tocopherol groups were self-assembled into choline-based BIL composed of lyposomic phospholipid bases containing hydrophilic and hydrophobic segments. These aggregates had a higher DNA density on the surface, biological stability as well as highly efficient cell internalization and showed the possibility of forming large polymersomes. [133] Another novel use of deep eutectic solvents based on protonated choline BILs is their application in the preparation of nanofibers via electrospinning. These fibers showed excellent hydrophilic characteristics and tunable morphology owing to aggregated structures. These have excellent potential for applications in tissue engineering. [134]

Conclusions and Future Outlook
Research on BILs has picked up significant steam in the last few years due to their multifarious, biocompatible, and green appeal in transformations and biomedical applications. The capacity to chemically transform them by employing their tunability allows for them to be incorporated into larger polymeric structures.
Our review focused on BILs and their biomedical engineering applications by discussing their properties relevant to the medical field. These ranged from antimicrobial and adhesive applications to power sources, devices, and tissue engineering. The review also described the various aspects of chemistry , and β-cyclodextrin (β-CD); b) schematic diagram of ionic gelation of IL ions with chitosan chains via different interactions, which finally forms chitosan-IL complexes. Reproduced with permission. [127] Copyright 2018, Royal Society of Chemistry.
www.advancedsciencenews.com www.advintellsyst.com and aggregated superstructures employed in specific biomedical vistas. Their ionic conduction capabilities, which are produced by their polymeric structures, rapidly expand their entry into electrochemistry, power storage, and devices. The pace of drug development research has been adversely affected by poor therapeutic efficiency and in vivo drug degradation. BILs can be a tool in the evolution of pharmaceutical growth, by advancing current drug delivery strategies that use BILs in liposomes, metallic nanoparticles, soft polymeric micellar vesicles, polymer dendrimers, polymer nanoparticles, or carbon nanostructures. BILs are being studied to improve drug stability in colloidal dispersions and enhance drug solubility. This is especially valuable for lipid-soluble drugs, as they are delivered to their targets via closed lipid vesicles composed of polymeric structures. For therapeutic solids, such strategies are also being studied to reduce aggregation, thus enhancing their viability.
A new frontier of BILs application is that of nucleic acid handling and delivery. Nucleic acids offer a window into gene function and to development of strategies for molecular medicine. This is predicated on the delivery of nucleic acid into eukaryotic cells, which suffer from limitations. An example of this would be the removal of RNA by BILs for prodrug platforms that are selfdelivering oligonucleotides. [120] Herein, such a complex structure would be capable of delivering oligonucleotides on its own with exceptional skin penetration, driving up the on-site bioavailability of the nucleotide. It was also proven by the experiment that the small interfering RNA (siRNA) showed cellular cytoplasmic localization. As an alternative control, BIL-mediated transfection of RNA caused the extraction of the nucleic acid, clearly establishing this as the preferable procedure. This approach is due to the advancement in both gene therapy and the functional targeting of malignancies.
Another breakthrough in this research is the employment of biopolymeric IL structures or copolymers, which are either based on ILs or polymers that have been functionalized by ILs. [120] DNA binding is strengthened by higher charge densities and the presence of hydroxyl groups, but this asset oftentimes comes with cytotoxicity. Contemporary structures based on polyethyleneimine and complexed with ammonium or phosphonium ILs can bypass this side effect, making it easier to apply them to biomedical treatments.
Research into BIL-based materials has evolved in recent years, proving that the materials are capable of more than acting as green solvents. Whether they are used on their own or are incorporated into APIs or complex therapeutics, their low immunogenicity, biocompatibility, biodegradability is broadening their range of applications. In research, their catalytic abilities have been applied for electrode structures. Advances due to their tailorability and easily retrofittable chemistry are encouraging their use in advanced diagnostics, sensors, field-effect transistor (FET) systems, wearable and implantable electronic devices, and regenerative smart tissue engineering. However, these emergent fields are still in the throes of early development. The future will have their potential being explored further with the marriage of intersectional multidisciplinary fields. Iman Noshadi is an assistant professor of bioengineering at the University of California Riverside. His research entails the development of biomaterials for biomedical application. His doctoral thesis from the University of Connecticut focused on developing poly(ionic liquid) porous materials for catalytic transformation of biomass. During his postdoctoral research at Harvard and MIT, he worked on biomaterials for regenerative biomedical applications. He has served as a faculty at Rowan university, where his group developed bioelectronics for regenerative medicine. Dr. Noshadi obtained his bachelor's from Shiraz University in Shiraz, Iran and his master's from University Technology, Malaysia.