Tunable Ionic Liquids: A Versatile Platform for Advanced Drug Delivery and Biomedical Applications

Abstract

This article provides a comprehensive exploration of ionic liquids (ILs) and their transformative potential in pharmaceutical sciences. Tailored for researchers and drug development professionals, it covers the foundational principles of IL tunability, from their modular cation-anion structures to their classification across generations. The scope extends to practical methodologies for formulating IL-based drug delivery systems, including self-assembled micelles and transdermal platforms. It addresses key challenges in optimization and biocompatibility, while presenting validation data and comparative analyses that underscore the advantages of ILs over conventional solvents. By synthesizing recent advances and clinical progress, this review serves as a strategic guide for harnessing ILs to overcome persistent drug delivery challenges.

The Building Blocks of Ionic Liquids: Understanding Modular Chemistry and Property Design

Ionic liquids (ILs) represent a unique class of organic salts that exist as liquids at relatively low temperatures, typically below 100 °C [1] [2]. Unlike conventional salts such as sodium chloride, which require extremely high temperatures to melt, ionic liquids possess inherently low melting points due to their molecular structure consisting of bulky, asymmetric organic cations paired with organic or inorganic anions [1] [3]. This structural composition prevents efficient packing into crystal lattices, thereby depressing their melting points and enabling liquid state at room temperature or moderately elevated temperatures [4].

The historical development of ionic liquids spans more than a century, beginning with Paul Walden's 1914 report on ethylammonium nitrate, which melts at 12°C [3] [4]. However, significant interest in these compounds emerged only in the late 20th century with the discovery of air- and water-stable varieties by Wilkes and Zaworotko in 1992 [3]. This breakthrough initiated what is now recognized as the "second generation" of ionic liquids, spurring extensive research into their unique properties and applications across diverse scientific and industrial fields [5] [3].

Ionic liquids are frequently described as "designer solvents" because their physicochemical properties can be precisely tailored for specific applications by selecting appropriate cation-anion combinations or incorporating functional groups into their molecular structure [6] [4]. This tunability, coupled with their exceptional properties, positions ionic liquids as transformative materials in areas ranging from sustainable chemistry to pharmaceutical development and energy technologies [5] [1].

Historical Evolution and Generational Classification

The development of ionic liquids has progressed through distinct generations, each marked by enhanced understanding and functional specialization. The classification system provides a framework for understanding how these materials have evolved from simple molten salts to sophisticated multifunctional compounds.

Table 1: Generational Classification of Ionic Liquids

Generation	Time Period	Key Characteristics	Example Applications	Limitations
First Generation	1914-1980s	Low melting point, high thermal stability, but sensitive to air and water [5] [3]	Electrolysis, electroplating [2]	Air and water sensitivity, low biodegradability [7]
Second Generation	1990s-present	Air and water stability, tunable physical and chemical properties [5] [3]	Green solvents, catalysis, separation processes [5] [2]	Variable toxicity, poor biodegradability for some types [7]
Third Generation	2000s-present	Biocompatibility, low toxicity, often derived from biological precursors [5] [7]	Pharmaceutical applications, biomedical uses [5] [2]	Limited commercial availability, higher cost
Fourth Generation	Emerging	Sustainability, biodegradability, multifunctionality [5]	Sustainable energy, green chemistry [5]	Still in developmental stages

The evolution from first to fourth-generation ionic liquids represents a paradigm shift from simply exploiting their low vapor pressure to deliberately designing their structure for specific functions and improved biocompatibility. Third-generation ILs, including Bio-ILs derived from natural sources like cholinium and amino acids, have been particularly significant for pharmaceutical applications due to their low toxicity and good biodegradability profile [7] [2]. The emerging fourth-generation ILs focus on sustainable design principles, emphasizing multifunctionality alongside environmental compatibility [5].

Fundamental Physicochemical Properties

Ionic liquids exhibit a remarkable combination of physical and chemical properties that distinguish them from conventional molecular solvents. These characteristics primarily stem from their ionic nature and complex molecular interactions.

Table 2: Key Physicochemical Properties of Ionic Liquids

Property	Typical Range/Value	Comparison with Conventional Solvents	Structural Dependence
Melting Point	<100°C (often below room temperature) [1]	Higher than molecular solvents but much lower than conventional salts	Governed by ion asymmetry, charge delocalization [3]
Vapor Pressure	Negligible to non-measurable [4]	Extremely low compared to volatile organic compounds	Ionic character prevents evaporation [3]
Thermal Stability	Up to 300-400°C for many ILs [4]	Superior to most molecular solvents	Determined by anion-cation combination [3]
Viscosity	20-40,000 cP [3]	Generally higher than water (∼1 cP)	Increases with alkyl chain length; affected by hydrogen bonding [3]
Ionic Conductivity	0.1-30 mS/cm [3]	Lower than aqueous electrolytes but sufficient for many applications	Depends on ion mobility and number of charge carriers [3]
Polarity	Wide range, from hydrophobic to highly hydrophilic [7]	Tunable across broader range than molecular solvents	Primarily determined by anion with cation modification [6]

The properties of ionic liquids are intimately connected to their structural components. Viscosity generally increases with longer alkyl chains on cations due to enhanced van der Waals interactions, while hydrogen bonding between cations and anions can further elevate viscosity [3]. Conductivity depends on both ion mobility and the number of charge carriers, with anions typically exerting a more significant influence than cations [3]. The polarity and hydrophilicity/hydrophobicity balance can be precisely adjusted through anion selection, with cations providing fine-tuning capability [6].

A distinctive feature of ionic liquids is their dynamic heterogeneity near the glass transition temperature. Research indicates that the size of cooperatively rearranging regions in ILs is considerably smaller than in their molecular counterparts with similar chemical structures, suggesting that electrostatic interactions significantly influence their dynamic behavior in the supercooled state [8].

Structural Diversity and Tunability

The versatility of ionic liquids originates from the vast combinatorial possibilities of cationic and anionic constituents. By selecting specific ion combinations and incorporating functional groups, researchers can design ILs with customized properties for specialized applications.

Common Ionic Liquid Structural Components

The most common cationic structures in ionic liquids include imidazolium, pyridinium, pyrrolidinium, ammonium, and phosphonium derivatives, often with varying alkyl chain substitutions that influence properties like hydrophobicity and viscosity [6] [2]. Anionic components range from simple halides to complex fluorinated species (e.g., [PF₆]⁻, [BF₄]⁻), amino acid derivatives, and carboxylates, with the anion typically exerting stronger influence on properties like polarity and hydrophilicity [6] [3].

Specialized subclasses of ionic liquids have emerged to address specific technological needs:

• Polymeric Ionic Liquids (PILs): ILs with polymerizable groups that form macromolecular structures with enhanced mechanical stability [6]
• Magnetic Ionic Liquids (MILs): Contain paramagnetic components for easy separation using magnetic fields [6]
• Zwitterionic Liquids (ZILs): Feature covalently tethered cations and anions within the same molecule [6]
• Active Pharmaceutical Ingredient ILs (API-ILs): Incorporate pharmaceutical agents as ionic constituents to improve drug properties [7]
• Surface Active ILs (SAILs): Amphiphilic structures that exhibit surfactant-like behavior [7]

This structural diversity enables precise optimization of ionic liquids for applications ranging from pharmaceutical formulations to energy storage systems, demonstrating the power of molecular design in creating task-specific materials.

Experimental Methodologies and Characterization Techniques

Quantitative Structure-Property Relationship (QSPR) Analysis

Predicting the behavior of ionic liquids is essential for their rational design. Quantitative Structure-Property Relationship (QSPR) modeling establishes mathematical relationships between structural descriptors of ILs and their physicochemical properties.

Experimental Protocol for Gas-Ionic Liquid Partition Coefficient Determination [9]:

• Sample Preparation: Prepare highly pure ionic liquid samples (>98% purity) and organic solute compounds. Ensure proper drying and storage to prevent moisture absorption.

• Inverse Gas-Liquid Chromatography (GLC):

  • Pack a chromatography column with the ionic liquid stationary phase
  • Use inert carrier gas (e.g., helium or nitrogen) with controlled flow rate
  • Inject volatile organic solute samples into the carrier gas stream
  • Measure retention times and volumes for each solute

• Partition Coefficient Calculation:

  • Calculate the gas-ionic liquid partition coefficient, K, using the formula: K = cIL/cG where cIL and cG represent solute concentrations in ionic liquid and gas phases, respectively [9]
  • Express results as log K values for linear modeling

• Molecular Descriptor Computation:

  • Generate theoretical molecular descriptors using software packages (e.g., Dragon)
  • Include constitutional, topological, geometrical, and electronic descriptors
  • Apply descriptor selection algorithms (e.g., replacement method, best multiple linear regression)

• Model Development:

  • Employ both linear (multiple linear regression) and non-linear (random forest) methods
  • Validate models using cross-validation and external validation sets
  • Interpret descriptor significance to identify key structural features influencing partitioning

QSPR Modeling Workflow

Key Research Reagents and Materials

Table 3: Essential Research Reagents for Ionic Liquid Studies

Reagent/Material	Function/Purpose	Example Specific Types
IL Cation Precursors	Provide cationic component of ILs	Imidazole, pyridine, pyrrolidine, tertiary amines [2]
IL Anion Sources	Provide anionic component of ILs	Metal salts (LiNTf₂, NaBF₄), acids (H₂SO₄, HPF₆) [2]
Organic Solutes	Probe molecules for property determination	n-Alkanes, aromatic compounds, functionalized organics [9]
Deuterated Solvents	NMR spectroscopy for structural verification	DMSO-d₆, CDCl₃, D₂O [3]
Chromatography Materials	Separation and analysis	GC columns, HPLC stationary phases [9] [6]
Drying Agents	Moisture removal for hydrophobic ILs	Molecular sieves, P₂O₅ [9]

Applications in Pharmaceutical and Biomedical Fields

Ionic liquids offer innovative solutions to persistent challenges in pharmaceutical development and biomedical applications. Their unique properties enable advances in drug delivery, formulation, and analysis.

Drug Solubilization and Bioavailability Enhancement

A significant challenge in pharmaceutical development involves the poor aqueous solubility of many drug candidates, which limits their bioavailability. Ionic liquids can dramatically improve drug solubility through multiple mechanisms:

• Direct Solubilization: ILs act as superior solvents for poorly water-soluble drugs, with studies demonstrating up to 120-fold solubility enhancement for compounds like ibuprofen [1]
• API-Ionic Liquid Formation: Converting active pharmaceutical ingredients into ionic liquid forms (API-ILs) can eliminate crystallinity issues that limit dissolution [7]
• Surface Active Properties: SAILs form micelles and colloidal structures that solubilize hydrophobic compounds above their critical micelle concentration [7]

The application of ILs in oral drug delivery is particularly promising, with the ability to overcome barriers associated with the Biopharmaceutics Classification System (BCS) Class II and IV drugs characterized by low solubility and variable permeability [7].

Drug Synthesis and Analysis

Ionic liquids serve multiple roles in pharmaceutical synthesis and analysis:

• Green Reaction Media: ILs replace volatile organic solvents in drug synthesis, facilitating reactions under milder conditions with improved yields and easier product separation [2]
• Catalysts: Functionalized ILs act as catalysts or co-catalysts in synthetic transformations, such as the esterification of curcumin achieving 98% yield in 15 minutes [2]
• Analytical Separations: IL-based stationary phases in chromatography improve separation efficiency for pharmaceutical compounds [6]
• Sample Preparation: ILs serve as effective extraction media in techniques like dispersive liquid-liquid microextraction (DLLME) for preconcentrating analytes from complex matrices [6]

Biomedical Applications

Beyond pharmaceutical formulations, ionic liquids find applications in various biomedical contexts:

• Antimicrobial Agents: Certain IL classes exhibit broad-spectrum antimicrobial activity, making them suitable for disinfectant formulations [2]
• Protein Stabilization: ILs can stabilize protein structure and function, enhancing the shelf-life of biopharmaceuticals [2]
• Drug Delivery Systems: ILs facilitate the development of novel nanocarriers for targeted drug delivery, including ionic liquid-based quantum dots and carbon nanotubes [1]

Ionic liquids represent a versatile class of materials whose unique properties stem from their ionic nature and structural diversity. As research progresses, the evolution of ionic liquids continues toward increasingly specialized applications, with fourth-generation ILs emphasizing sustainability, biodegradability, and multifunctionality [5].

Future developments will likely focus on several key areas:

• Sustainable Design: Increased emphasis on bio-derived ionic liquids with reduced environmental impact and improved biocompatibility [5] [7]
• Pharmaceutical Innovations: Expansion of API-IL technologies to address formulation challenges for increasingly complex drug molecules [7] [2]
• Energy Applications: Development of ILs for advanced energy storage systems, including batteries, supercapacitors, and thermal energy storage [5] [4]
• Predictive Modeling: Enhanced computational approaches for accurate prediction of IL properties, reducing the need for extensive experimental screening [9] [3]

The extraordinary versatility of ionic liquids, coupled with their tunable properties, ensures their continued importance as enabling materials across scientific disciplines. As fundamental understanding of their structure-property relationships deepens, ionic liquids will undoubtedly play an increasingly vital role in developing sustainable technological solutions to contemporary challenges in pharmaceuticals, energy, and materials science.

Ionic liquids (ILs), a class of materials typically defined as salts melting below 100 °C, have emerged as a transformative platform in materials science and chemical engineering. Their evolution is categorized into distinct generations: first-generation ILs as green solvents; second-generation ILs designed for specific applications in catalysis and electrochemistry; third-generation ILs incorporating bio-derived and task-specific functionalities; and fourth-generation ILs focusing on sustainability and multifunctionality [5]. The defining characteristic of all these generations is their inherent modularity—the ability to tailor their physicochemical properties by the rational selection of cation-anion combinations.

This principle of modular design extends beyond conventional ionic liquids to advanced materials, including ionic liquid crystals (ILCs) and complex multi-anion inorganic solids [10] [11]. In all cases, the careful selection of ionic components allows researchers to pre-program key material properties. This guide explores the fundamental mechanisms by which cation-anion pairings dictate physicochemical behavior and provides researchers with the methodological tools to harness this principle for designing functional materials.

Fundamental Principles of Ionic Modularity

The "Designer Solvent" Paradigm

The structure of ionic liquids consists typically of a large, asymmetric organic cation and a smaller inorganic or organic anion [1]. This architectural asymmetry is fundamental to their low melting points and liquid state at room temperature. The "designer solvent" concept arises from the ability to independently modify the cation or anion, or to incorporate specific functional groups, to achieve targeted physicochemical properties [1].

The properties arising from cation-anion combinations can be broadly categorized:

• Properties primarily determined by the anion: Thermal stability, miscibility with other solvents, and hydrogen bonding capacity [1].
• Properties primarily influenced by the cation: Viscosity, surface tension, and density, often modulated by varying the length and branching of alkyl chains attached to the cationic core [1].
• Properties arising from synergistic interactions: Polarity, electrochemical window, and solvation behavior, which emerge from the specific pairing of ions [1].

Orthogonality in Ion-Driven Self-Assembly

A sophisticated manifestation of the modular principle is found in orthogonal self-assembly, where cation and anion binding sites are pre-programmed to operate independently. As demonstrated in subcomponent self-assembly, cation coordination and anion recognition can function as orthogonal structure-directing elements [12].

This orthogonality enables a modular build-up of structure in multi-ion assemblies. For instance, when sites for anions are pre-programmed (e.g., using cyanostar macrocycles for anion coordination) and combined with cation-binding ligands, structure can be built up predictably via either cation-anion or anion-cation pathways [12]. This approach allows for the isolation of intermediates and the modular construction of complex architectures by sequentially adding structure-directing ions, showcasing the power of orthogonal design in supramolecular chemistry [12].

Key Physicochemical Properties and Their Modulation

Property Tuning Through Cation-Anion Selection

Table 1: Modulation of Key Physicochemical Properties through Cation-Anion Selection

Property	Influencing Factors	Modulation Strategy	Typical Range
Viscosity	Cation alkyl chain length, anion size, hydrogen bonding	Increase alkyl chain length; use symmetric anions to increase viscosity; use weakly coordinating anions to decrease	High (significantly greater than conventional solvents) [13]
Conductivity	Ion mobility, ion concentration, viscosity	Use small anions and cations; reduce viscosity; design for weak ion pairing	Behave as strong electrolytes [13]
Vapor Pressure	Cohesive energy, intermolecular forces	Use ions with delocalized charges and asymmetric shapes to minimize	Negligible under normal conditions [13]
Thermal Stability	Anion nucleophilicity, cation stability	Use fluorinated anions (e.g., BF₄⁻, PF₆⁻); use aromatic cations (e.g., imidazolium)	High thermal stability [1]
Solvation Capacity	Polarity, hydrogen bond basicity/anion, functional groups	Tune anion for hydrogen bond basicity; add functional groups to cation	Can dissolve polar, non-polar, polymeric, and biological molecules [1]
Electrochemical Window	Anion oxidation stability, cation reduction stability	Use inert anions (e.g., [NTf₂]⁻); use stable cations (e.g., phosphonium)	Large electrochemical windows (>4V) [1]

Advanced Modularity in Multi-Anion Materials

Beyond single cation-anion pairs, the modular principle extends to multiple anion materials, where different anions occupy distinct structural roles. In the van der Waals material Bi₄O₄SeCl₂, the presence of three anions (oxide, selenide, chloride) creates a 1:1 superlattice of structural units from BiOCl and Bi₂O₂Se [11].

In this system:

• Chloride acts as a terminal ligand, producing van der Waals gaps that enable exfoliation
• Selenide serves as a bridging ligand, connecting Bi₂O₂ layers and defining electronic properties
• Oxide remains as a structural component within the cationic layers [11]

This strategic combination of terminal and bridging anions retains the desirable electronic properties of Bi₂O₂Se while introducing the exfoliability of BiOCl, creating a new van der Waals material with high electronic mobility [11]. This demonstrates how advanced modular design with multiple anions can yield materials with hybrid functionalities.

Experimental Methodologies for Property Characterization

Viscosity Measurements

Principle: Viscosity of ionic liquids is significantly higher than conventional molecular solvents due to strong Coulombic interactions and hydrogen bonding [13]. The measurement compares flow behavior against reference standards.

Protocol:

• Use a calibrated Ostwald viscometer or rotational rheometer
• Maintain temperature control with ±0.1°C precision
• Measure flow time for reference solvent (e.g., water) and IL sample
• Calculate kinematic viscosity: η = kρt, where k is viscometer constant, ρ is density, t is flow time
• Perform measurements in triplicate across temperature ranges (e.g., 20-80°C)

Data Interpretation: Imidazolium-based ILs typically show higher viscosity than molecular solvents. Viscosity decreases with increasing temperature and with anion size (e.g., [NTf₂]⁻ < [PF₆]⁻ < [BF₄]⁻) [13].

Conductivity Measurements

Principle: Ionic liquids behave as strong electrolytes, with conductivity dependent on ion mobility, which is inversely related to viscosity [13].

Protocol:

• Use a conductivity cell with platinum electrodes
• Apply alternating current to prevent electrolysis
• Calibrate with standard KCl solutions (0.01 M, 0.1 M, 1.0 M)
• Measure cell constant (K) using relationship: K = κₛₜRₛₜ, where κₛₜ is standard conductivity, Rₛₜ is measured resistance
• Measure resistance of IL sample and calculate conductivity: κ = K/R
• Maintain constant temperature during measurements

Data Interpretation: Conductivity trends generally inverse to viscosity. Larger ions typically show lower conductivity due to reduced mobility [13].

Vapor Pressure Characterization

Principle: Ionic liquids possess negligible vapor pressure under normal conditions due to strong Coulombic forces, distinguishing them from molecular solvents [13].

Protocol:

• Use an isoteniscope or static method apparatus
• Purge IL sample to remove volatile impurities and dissolved gases
• Apply vacuum to the system
• Measure pressure at various temperatures
• Compare with molecular solvents (e.g., water, ethanol)

Data Interpretation: ILs show no measurable vapor pressure at room temperature, unlike molecular solvents which obey the Clausius-Clapeyron equation [13]. This property is crucial for their application as green solvents.

Diagram 1: Workflow for characterizing ionic liquid properties

Case Studies in Modular Design

Orthogonal Cation-Anion Self-Assembly

A sophisticated demonstration of the modular principle involves combining cyanostar macrocycles (anion receptors) with metal-binding ligands for orthogonal self-assembly [12]. This system enables the independent programming of cation and anion binding sites.

Experimental Protocol:

• Synthesis of mono-substituted cyanostar (NH₂-CS): Functionalize cyanostar macrocycle with 4-aminophenyl group to enable imine formation
• Preparation of metal complexes: Use {POPCu(MeCN)₂}+ or {PPh₃Au}+ as metal precursors with labile ligands
• Imine condensation: React NH₂-CS with fluoro-substituted picolinaldehyde (F-PyCHO) in presence of metal ions
• Isolation of intermediates: Use non-coordinating counterions (BArF₄⁻, NTf₂⁻, TBA⁺) to isolate cationic [LCu-CS]+ or anionic [NH₂CS-BF₄-NH₂CS]⁻ intermediates
• Stepwise assembly: Combine intermediates to form final [LM-CS-X-CS-ML]+ architecture

Key Findings:

• Assembly proceeds via discrete steps with isolable intermediates
• Different products can be prepared modularly using Au⁺ and ClO₄⁻ ions
• First example of subcomponent self-assembly with Au(I) [12]
• Pre-programmed cation and anion binding sites serve as orthogonal interactions

Diagram 2: Orthogonal cation-anion self-assembly pathways

Dicationic Ionic Liquid Crystals

Dicationic ionic liquid crystals (DILCs) represent another manifestation of modular design, featuring two single-charged ions covalently linked by a spacer, offering enhanced tunability [10].

System Characteristics:

• Enhanced thermal stability compared to monocationic analogs
• Variable spacer length and composition modulates mesophase behavior
• Two long alkyl chains enhance rod-shaped character and promote mesophase formation
• Layered smectic phases stabilized by electrostatic interactions in ionic sublayers and van der Waals forces in hydrophobic domains [10]

Experimental Analysis (NMR):

• Director alignment: Slowly cool (<1°C/min) from isotropic phase in strong magnetic field (≥10 T)
• ¹³C NMR measurements: Perform at static conditions without magic angle spinning
• Order parameter determination: Analyze dipolar couplings to determine conformational parameters and orientational order
• Comparative analysis: Compare with monocationic analogs to elucidate structural effects [10]

Key Findings:

• Dynamic spacer effectively "decouples" motion of two ionic moieties
• Bond order parameters in side chains lower than double-chain monocationic analogs
• Layered structure stabilized despite less ordered dications due to increased interaction energy in polar domain [10]

The Scientist's Toolkit: Research Reagent Solutions

Table 2: Essential Research Reagents for Ionic Materials Development

Reagent/Material	Function/Application	Key Characteristics	Examples
Cyanostar Macrocycles	Pre-programmed anion binding sites	Strong anion coordination, forms 2:1 sandwich complexes with bridging anions	Mono-substituted cyanostar (NH₂-CS) for imine condensation [12]
Imidazolium Salts	Versatile cationic platform	Tunable alkyl chain length, forms various mesophases, good thermal stability	3,3'-(1,6-hexanediyl)bis(1-dodecylimidazolium) for DILCs [10]
Non-Coordinating Ions	Isolating intermediates, weak ion pairing	Poor coordination to both metals and receptors, enables stepwise assembly	BArF₄⁻, NTf₂⁻ anions; TBA⁺ cations [12]
Metal Precursors	Cation coordination in self-assembly	Labile ligands, specific coordination geometries	{POPCu(MeCN)₂}+ (Cu⁺ source), {PPh₃Au}+ (Au⁺ source) [12]
Multiple Anion Precursors	Designing complex inorganic materials	Provide distinct terminal and bridging functionalities	BiOCl, Bi₂O₂Se for creating Bi₄O₄SeCl₂ superlattices [11]

The modular principle governing cation-anion combinations represents a powerful paradigm in materials design. From the fundamental level of selecting ion pairs to dictate basic physicochemical properties, to the sophisticated engineering of orthogonal binding sites for complex self-assembly, this principle enables unprecedented control over material behavior.

The experimental methodologies and case studies presented provide researchers with both the theoretical framework and practical tools to exploit this modularity. As the field advances, the integration of computational design with synthetic methodology will further enhance our ability to predict and optimize cation-anion combinations for targeted applications. The growing understanding of multi-anion materials and orthogonal self-assembly pathways points toward an increasingly sophisticated application of the modular principle in developing next-generation functional materials.

Ionic liquids (ILs), a class of materials defined as salts with a melting point below 100 °C, have emerged as a transformative force in chemical research and application [14]. Their significance stems not from a single property, but from a unique combination of characteristics—including low volatility, high thermal stability, and tunable solubility—that can be precisely engineered for specific tasks [5]. This tunability is the cornerstone of IL research, allowing scientists to design salts with bespoke physicochemical and biological properties by selecting and modifying the cationic and anionic components [15] [16]. This capacity for molecular-level design has propelled ILs through a distinct evolutionary pathway, characterized by four generations, each expanding their capabilities from fundamental electrochemistry to advanced biomedical and sustainable technologies [5] [17].

The following diagram illustrates this evolutionary pathway, showing the key focus and applications of each generation of ionic liquids.

The Historical Pathway of Ionic Liquid Generations

The First Generation: Foundation and Early Applications

The earliest ionic liquids were discovered independently by several research groups, with the first stable room-temperature ionic liquid, ethylammonium nitrate ([EtNH3][NO3], m.p. 12 °C), being reported by Paul Walden in 1914 [18] [14]. However, the potential of this discovery remained largely unexplored for decades. Modern interest was rekindled in the mid-20th century with the work of Hurley and Weir, who used mixtures of 1-ethylpyridinium bromide and aluminium chloride for the electrodeposition of metals [18]. A significant breakthrough came in the 1980s when John Wilkes' group introduced 1,3-dialkylimidazolium cations, which offered improved transport properties and stability, establishing the archetype for subsequent IL research [18].

The primary characteristic of first-generation ILs was their utility as green solvents with unique physical properties, notably for electroplating and as electrolytes [5]. These chloroaluminate-based ILs were highly sensitive to water and air, requiring specialized handling in inert atmospheres, which limited their widespread adoption [18]. A major drawback was their low biodegradability and significant toxicity to aquatic environments [15].

The Second Generation: Task-Specific Design

Second-generation ILs emerged as researchers sought more stable and designable materials. A pivotal moment was the 1992 work by Wilkes and Zawarotko, who synthesized ILs with 'neutral' weakly coordinating anions like hexafluorophosphate (PF₆⁻) and tetrafluoroborate (BF₄⁻) [14]. This innovation yielded ILs that were stable in air and water, dramatically broadening their application potential [15].

This generation shifted the focus from mere solvent properties to task-specific functionality [5]. By systematically adjusting the alkyl chain lengths on cations or pairing them with different anions, scientists could fine-tune physical properties such as melting point, viscosity, hydrophilicity, and solubility for specific applications [15]. This led to their use in homogeneous and biphasic catalysis, lubricants, and as advanced electrolytes in batteries and supercapacitors [5] [17].

The Third Generation: The Biocompatibility Revolution

The third generation marked a paradigm shift towards biocompatibility and bio-derived components [5]. Instead of merely reducing toxicity, researchers began constructing ILs from ions with known biological activity or from natural, renewable sources [16]. Common cations included choline—a vitamin essential for human health and classified as "Generally Regarded as Safe" (GRAS) by the FDA—while anions were derived from amino acids, fatty acids, and carboxylic acids [19].

These ILs were designed not just to be less toxic, but to interact with biological systems in a predictable, beneficial way [16]. This opened the door to applications in pharmaceutics and biomedicine, including their use to enhance drug solubility, improve formulation stability, and act as antimicrobial agents or drug delivery vehicles [5] [15] [19]. Their low cost and simplicity of synthesis from natural materials further enhanced their appeal [19].

The Fourth Generation: Sustainable Multifunctionality

The most recent evolution, fourth-generation ILs, integrates the functionalities of previous generations with an overarching emphasis on sustainability, biodegradability, and multifunctionality [5] [17]. This generation represents a holistic approach where ILs are engineered to be inherently green throughout their lifecycle, from synthesis to disposal.

The focus is on developing smart, biodegradable, and recyclable materials with tailored functionalities for next-generation applications [5]. This includes ILs designed for precision medicine, where they can selectively target diseased cells, and for sustainable industrial processes like CO₂ capture and utilization [5]. Fourth-generation ILs are poised to drive advancements in green chemistry, renewable energy, and biocompatible technologies, positioning them as key enablers of a sustainable and technologically advanced future [5].

Table 1: Key Characteristics of Ionic Liquid Generations

Generation	Primary Focus	Example Components	Key Advantages	Inherent Limitations
First	Green Solvents [5]	Chloroaluminates; Imidazolium/Pyridinium cations [15] [18]	Low vapor pressure; High thermal stability [5]	Water/air sensitivity; High toxicity; Poor biodegradability [15] [18]
Second	Task-Specific Design [5]	Imidazolium/Ammonium cations; [PF₆]⁻, [BF₄]⁻ anions [15] [14]	Air/water stability; Tunable physicochemical properties [15]	Variable toxicity; Not inherently biodegradable [15]
Third	Biocompatibility [5]	Choline cations; Amino acid, fatty acid anions [15] [19]	Low toxicity; Biodegradable; Biologically active [15] [19]	Complex synthesis; Limited property range from natural ions [19]
Fourth	Sustainable Multifunctionality [5] [17]	Biodegradable cations & anions; Smart functional groups	Full lifecycle sustainability; Smart & recyclable materials [5]	Early stage of development; High cost of some components

Experimental Protocols in Ionic Liquid Research

Synthesis of a Third-Generation Choline-Amino Acid IL

The synthesis of biocompatible ILs, such as choline-amino acid salts, is a fundamental protocol in modern IL research [19]. The following workflow details the neutralization method, a common and straightforward approach.

Detailed Methodology:

• Reaction Setup: An aqueous solution of choline hydroxide is placed in a round-bottom flask. A slight excess (e.g., 1.05 equivalents) of the chosen amino acid (e.g., glycine, alanine, proline) is added to the stirring solution to ensure complete conversion of the choline base [19].
• Reaction Progression: The reaction mixture is stirred for 12 to 24 hours at room temperature or at a mildly elevated temperature (e.g., 40 °C) to ensure complete salt formation [19].
• Purification: The water and other volatile components are removed from the reaction mixture using rotary evaporation under reduced pressure.
• Drying: The resulting viscous liquid is placed under a high vacuum (e.g., < 0.1 mbar) for an extended period, often 24 hours at 50 °C, to eliminate any residual water or solvents, yielding the final pure ionic liquid [19].
• Characterization: The structure and purity of the IL are confirmed by nuclear magnetic resonance (NMR) spectroscopy. Its thermal stability is assessed using thermogravimetric analysis (TGA), and other properties can be analyzed with Fourier-transform infrared (FTIR) spectroscopy and differential scanning calorimetry (DSC) [19].

Application Protocol: Using ILs as Solvents and Catalysts in Drug Synthesis

A representative experiment demonstrating the dual role of ILs as solvent and catalyst is the synthesis of curcumin diacetate, as investigated by Marcin et al. [15].

Detailed Methodology:

• Reaction Setup: The reaction is set up in a controlled atmosphere. 1-Butyl-3-methylimidazolium bis(trifluoromethylsulfonyl)imide ([C₄C₁im][N(Tf)₂]) is placed in a reaction vessel. Curcumin and acetic anhydride are added to the IL.
• Esterification: The reaction mixture is stirred under optimized conditions for 15 minutes. The IL acts as both the solvent medium and a catalyst, significantly accelerating the reaction compared to conventional solvents [15].
• Product Separation: After the reaction is complete, depressurized filtration is performed to separate the ionic liquid from the solid reaction mixture [15].
• IL Recycling: The filtrate containing the ionic liquid is washed three times with ethyl acetate to dissolve and remove any residual organic products. The recovered ionic liquid is then dried in a vacuum dryer for 24 hours at 50 °C in preparation for reuse in subsequent reaction cycles. Studies have shown that ILs like [C₆C₁im][N(Tf)₂] can be recycled three times without a significant loss in catalytic activity [15].

The Scientist's Toolkit: Essential Research Reagents

Table 2: Key Reagent Solutions in Ionic Liquid Research

Reagent Category	Specific Examples	Function in Research
Cationic Precursors	1-Methylimidazole; Choline chloride; Alkylpyridines; Tetraalkylammonium halides	Serves as the foundational starting material for synthesizing the cationic component of ILs. Alkyl chain length can be varied to tune properties [15] [19].
Anion Sources	Amino acids (Glycine, Alanine); Fatty acids; Hexafluorophosphoric acid (HPF₆); Lithium bis(trifluoromethylsulfonyl)imide (LiNTf₂)	Used in metathesis or neutralization reactions to introduce the anionic component. Choice of anion heavily influences solubility, toxicity, and catalytic activity [15] [19].
Biocompatible IL Kits	Choline-Glycine; Choline-Octanoate; 1-Benzyl-3-methylimidazolium Dicyanamide	Ready-made or easily synthesized ILs used as benchmarks for evaluating biological activity, drug solubility enhancement, and antimicrobial efficacy [16] [19].
Characterization Standards	Deuterated solvents (DMSO-d6, CDCl3); Analytical standards for HPLC/LC-MS	Essential for confirming IL structure (NMR), assessing purity (Chromatography), and quantifying performance in applications like drug analysis [15].

The evolution of ionic liquids from simple, specialized solvents to sophisticated, sustainable, and multifunctional materials illustrates the power of tunable chemistry. This journey, segmented into four distinct generations, has seen the focus shift from fundamental physical properties to task-specific functionality, then to inherent biocompatibility, and finally to a holistic sustainability mandate. Today, fourth-generation ILs are paving the way for advancements in precision medicine, where they can be designed for selective anticancer or antimicrobial activity, and in next-generation energy storage systems [5] [16]. Furthermore, their role in creating sustainable industrial processes, such as biodegradable lubricants and efficient CO₂ capture systems, underscores their potential as key enablers of a green technological future [5] [17]. As research progresses, the exploration of ILs within this generational framework promises to yield even smarter, more integrated materials capable of addressing some of the most complex challenges in science and industry.

Ionic Liquids (ILs), often described as "designer solvents," are a class of materials typically composed of organic cations and organic or inorganic anions with melting points below 100°C [20] [6]. Their most defining characteristic is the tunability of their physicochemical properties, which can be precisely adjusted by selecting different cation-anion combinations or by incorporating functional groups into their molecular structures [20] [6]. This guide focuses on four cornerstone tunable properties—solubility, viscosity, thermal stability, and hydrogen-bonding capacity—that are critical for tailoring ILs for advanced applications in drug development, energy storage, and separation sciences [5] [20] [21]. A fundamental understanding of these properties enables researchers to systematically design ILs with optimized performance for specific technological challenges.

The Four Key Tunable Properties

Solubility

The solubility of Ionic Liquids, and their capacity to dissolve other substances, is primarily governed by the selection of ions. The nature of the anion often plays a dominant role in determining the hydrophilicity or hydrophobicity of the IL, while the cation structure can influence interactions with organic molecules [20] [6]. This tunability allows ILs to serve as powerful solubility enhancers for poorly soluble drugs, thereby addressing a major challenge in pharmaceutical development [20]. By optimizing anion-cation pairs, ILs can be engineered to dissolve a wide range of materials, from pharmaceuticals to biopolymers, making them versatile solvents for numerous applications [5].

Table 1: Strategies for Tuning Ionic Liquid Solubility

Target Solubility	Cation Modification	Anion Selection	Key Effect
Hydrophilicity	Short alkyl chains (e.g., methyl, ethyl)	Chloride (Cl⁻), Bromide (Br⁻), Nitrate ([NO₃]⁻)	Increases water miscibility and polarity [20]
Hydrophobicity	Long alkyl chains (e.g., hexyl, octyl)	Hexafluorophosphate ([PF₆]⁻), Bis(trifluoromethylsulfonyl)imide ([NTf₂]⁻)	Creates water-immiscible phases for extractions [6]
Drug Solubility Enhancement	Tunable to prevent drug crystallization	Hydrogen bond-accepting anions (e.g., [CH₃COO]⁻)	Disrupts drug crystal lattice, improving bioavailability [20]
Polymer Solubility	Imidazolium-based cations	Chloride (Cl⁻), Acetate ([CH₃COO]⁻)	Efficiently dissolves biopolymers like cellulose [5]

Viscosity

Viscosity is a critical transport property for Ionic Liquids, influencing mass transfer, diffusion rates, and overall process efficiency. ILs tend to be more viscous than conventional organic solvents, primarily due to strong electrostatic forces and hydrogen bonding between ions, which impede fluid flow [21]. Key factors for tuning viscosity include the size and flexibility of the ions: bulkier, more asymmetric ions typically lead to higher viscosities because they cannot pack efficiently, while shorter alkyl chains on cations generally result in lower viscosities [20] [21]. The anion type is another powerful control parameter; for instance, ions with strong hydrogen-bond-accepting abilities can reduce intermolecular attractions and lower viscosity [21].

Table 2: Factors Influencing Ionic Liquid Viscosity

Factor	Impact on Viscosity	Molecular-Level Rationale	Example
Alkyl Chain Length	Increases with longer chains	Enhanced van der Waals interactions between chains [20] [21]	[C₂MIm][NTf₂] vs [C₈MIm][NTf₂]
Anion Type	Varies significantly with anion identity	Modifies strength of Coulombic & hydrogen-bonding interactions [21]	[C₄MIm][Cl] (high) vs [C₄MIm][NTf₂] (low)
Temperature	Decreases exponentially with temperature	Increased thermal energy overcomes intermolecular forces [21]	Universal behavior for ILs
Hydrogen Bonding	Stronger H-bonding increases viscosity	Creates a cohesive, interconnected network between ions [21]	ILs with [CF₃COO]⁻ vs [NTf₂]⁻

Thermal Stability

The thermal stability of Ionic Liquids, defined by their resistance to decomposition at high temperatures, is a key advantage over volatile organic solvents. This property is heavily dependent on the strength of the heterocyclic cation and the nucleophilicity of the anion [5] [21]. ILs with aromatic cations like imidazolium and triazolium generally exhibit high thermal stability, while anions that are weakly coordinating and resistant to decomposition (e.g., [NTf₂]⁻) further enhance stability [21]. This robust thermal profile allows ILs to be employed in high-temperature processes, including as green propellants (Energetic ILs) and as stable electrolytes in energy storage devices [5] [21].

Table 3: Thermal Stability of Ionic Liquids by Ion Type

Ion Type	Specific Example	Typical Decomposition Onset Range (°C)	Influencing Factors
Imidazolium Cations	1-Butyl-3-methylimidazolium ([C₄MIm]⁺)	400 - 450 °C [21]	Anion nucleophilicity; alkyl chain length
Triazolium Cations	1,2,4-Triazolium-based cations	> 400 °C [21]	Cation structure; functional groups
Phosphonium Cations	Trihexyl(tetradecyl)phosphonium ([P₆₆₆₁₄]⁺)	Often > 400 °C [5]	Strength of C-P bonds; anion stability
Stable Anions	Bis(trifluoromethylsulfonyl)imide ([NTf₂]⁻)	Contributes to high stability [21]	Weak coordination; strong chemical bonds

Hydrogen-Bonding Capacity

Hydrogen-bonding capacity is a fundamental property that dictates the solvation character and miscibility of Ionic Liquids. ILs can act as both hydrogen bond donors (via C–H groups on the cation, most notably the acidic proton on the C2 position of imidazolium) and hydrogen bond acceptors (primarily through the anion) [6]. This dual nature is a powerful tool for tuning IL interactions with solute molecules. The strength and extent of hydrogen bonding can be modulated by choosing anions with different hydrogen bond acceptance strengths (e.g., acetate [CH₃COO]⁻ is a strong acceptor, while [NTf₂]⁻ is weak) and by modifying the cation structure to enhance or reduce its donor ability [20] [6]. This property is crucial for applications like stabilizing biomolecules in pharmaceuticals or facilitating the extraction of specific compounds [20].

Diagram 1: A strategy map for tuning the hydrogen-bonding capacity of ionic liquids through anion selection and cation modification.

Experimental Protocols for Property Characterization

Protocol for Solubility Enhancement Studies

This protocol outlines a method to evaluate the ability of ILs to enhance the solubility of poorly soluble Active Pharmaceutical Ingredients (APIs), a key application in drug development [20].

• IL Synthesis/Selection: Synthesize or procure the target ILs. A common starting point is imidazolium-based ILs with anions like acetate or chloride, known for their good solvating power [20]. Purify the ILs to remove volatile impurities and water.
• Sample Preparation: Prepare a series of solutions with a fixed concentration of the IL in a suitable solvent (e.g., water or buffer). Into each solution, add an excess of the poorly soluble API.
• Equilibration: Agitate the mixtures continuously using a vortex mixer or an orbital shaker for a defined period (e.g., 24-48 hours) at a constant temperature (e.g., 37°C) to reach equilibrium [20].
• Separation: Centrifuge the samples at high speed (e.g., 10,000 rpm for 10 minutes) to separate the undissolved API from the saturated solution.
• Analysis: Dilute the supernatant appropriately and analyze the concentration of the dissolved API using a calibrated High-Performance Liquid Chromatography (HPLC) system with UV detection [20]. Compare the solubility in the IL solution against the solubility in a pure buffer to calculate the fold-increase.

Protocol for Thermogravimetric Analysis (TGA)

TGA is the standard method for determining the thermal decomposition onset temperature of Ionic Liquids, providing a quantitative measure of their thermal stability [21].

• Instrument Calibration: Calibrate the TGA instrument for temperature using magnetic standards (e.g., Nickel or Perkalloy).
• Sample Loading: Place a small, precisely weighed sample of the IL (typically 5-10 mg) into an open, inert crucible (e.g., alumina).
• Experimental Parameters: Run the analysis under an inert nitrogen atmosphere (flow rate ~ 50 mL/min) to prevent oxidative degradation. Use a controlled heating rate, commonly 10°C per minute, from room temperature to a high temperature (e.g., 500-600°C).
• Data Analysis: Plot the percentage weight loss as a function of temperature. The thermal decomposition onset temperature (Tₒₙₛₑₜ) is determined as the temperature at which a predefined weight loss (e.g., 1% or 5%) occurs, or alternatively, by the intersection of tangents to the baseline and the decomposition curve [21].

Protocol for Dispersive Liquid-Liquid Microextraction (DLLME)

This protocol utilizes the tunable solubility and hydrogen-bonding capacity of ILs for efficient sample preparation and pre-concentration of analytes [6].

• Solution Preparation: Prepare the aqueous sample solution containing the target analytes. Select a water-immiscible IL (e.g., [C₆MIm][PF₆]) as the extraction solvent.
• Dispersion: Rapidly inject the IL (as the extraction solvent) using a syringe into the aqueous sample. This creates a cloudy solution consisting of fine droplets of IL dispersed throughout the aqueous phase, which provides a large surface area for extraction [6].
• Equilibration: Gently mix the solution to allow the analytes to partition from the aqueous sample into the IL droplets. This step can be assisted by vortexing or ultrasonication.
• Phase Separation: Centrifuge the mixture for a few minutes to break the emulsion and sediment the dense IL phase at the bottom of the tube. . Analysis: Carefully remove the aqueous phase with a syringe. The sedimented IL phase, now enriched with the target analytes, can be redissolved in a compatible solvent and analyzed via HPLC, GC, or other instrumental methods [6].

Diagram 2: A generalized experimental workflow for ionic liquid research, from synthesis and property characterization to functional application.

The Scientist's Toolkit: Essential Research Reagents & Materials

Table 4: Key Reagents and Materials for Ionic Liquid Research

Reagent/Material	Function/Application	Relevance to Tunable Properties
1-Methylimidazole	A common precursor for synthesizing imidazolium-based IL cations [20].	The foundational building block; alkyl chain length can be varied to tune viscosity & hydrophobicity.
Alkyl Halides (e.g., Bromobutane)	Used to quaternize the nitrogen atom of cations like imidazole, forming the IL structure [20].	The alkyl chain (R-group) length is a primary handle for tuning viscosity & lipophilicity.
Lithium Bis(trifluoromethylsulfonyl)imide (Li[NTf₂])	A common source for the [NTf₂]⁻ anion via metathesis reactions.	Imparts hydrophobicity, low viscosity, and high thermal/electrochemical stability [21].
Sodium Acetate (Na[CH₃COO])	A source for the acetate anion for metathesis.	Provides strong hydrogen-bond acceptance, enhancing solubility for pharmaceutical APIs [20].
Activated Charcoal	Used for purification of synthesized ILs to remove colored impurities.	Essential for achieving high-purity ILs, which ensures accurate and reproducible property data.
Deuterated Solvents (e.g., DMSO-d₆)	Solvents for Nuclear Magnetic Resonance (NMR) characterization.	Critical for confirming IL chemical structure and purity, and for studying H-bonding via NMR spectroscopy.

Ionic liquids (ILs), a class of materials defined as salts with melting points below 100°C, have emerged as transformative tools in pharmaceutical research and development. Their properties, including low volatility, high thermal stability, and tunable solubility, are governed by the selection of their cationic and anionic components. The evolution of ILs is categorized into generations, with later generations emphasizing biocompatibility and multifunctionality [5]. This versatility allows for the rational design of ILs to overcome persistent challenges in drug development. Within this context, three major classes have garnered significant attention for their distinct advantages: imidazolium-based ILs, valued for their versatility and efficiency; cholinium-based ILs, recognized for their exceptional biocompatibility; and Active Pharmaceutical Ingredient ILs (API-ILs), which represent a paradigm shift in drug formulation. This whitepaper provides an in-depth technical guide to these core classes, detailing their properties, applications, and experimental methodologies, framed within the broader thesis of harnessing tunable IL properties for advanced pharmaceutical applications.

Ionic liquids are often described as "designer solvents" because their physicochemical characteristics can be finely adjusted through a meticulous selection and functionalization of the cation-anion pair. This tunability is the foundational principle enabling their diverse applications in pharmaceuticals. The evolution of ILs can be understood through their generational development, which reflects a growing emphasis on sustainability and biological integration [5].

• First-Generation ILs: Early ILs, such as those based on dialkylimidazolium cations with metal halide anions, were developed primarily for electrochemical applications. Their properties, including high thermal stability and low melting points, made them suitable as functional solvents, though they often exhibited low biodegradability and high toxicity [15].
• Second-Generation ILs: This class expanded the repertoire of anions to include species like tetrafluoroborate (BF₄⁻) and hexafluorophosphate (PF₆⁻), resulting in ILs stable in air and water. Their key advancement is the ability to customize properties like melting point, viscosity, and hydrophilicity for specific functional materials and processes [15] [5].
• Third-Generation ILs: Marking a significant turn towards biocompatibility, this generation incorporates ions from natural or metabolic sources, such as cholinium cations and amino acid anions. These ILs are characterized by low toxicity and good biodegradability, making them suitable for biomedical applications like drug delivery and stabilization of biologics [15] [22].
• Fourth-Generation ILs: An emerging frontier focuses on sustainable, biodegradable, and multifunctional ILs that integrate smart capabilities for next-generation applications in precision medicine and green chemistry [5].

The following diagram illustrates the logical relationship between the tunable design of ILs and their resulting properties and applications in the pharmaceutical field.

Major Ionic Liquid Classes in Pharma

Imidazolium-Based ILs

Imidazolium cations are one of the most extensively studied cores in IL chemistry. Their popularity stems from a combination of structural adaptability, good thermal stability, and broad utility as solvents and catalysts.

• Structure and Properties: The core structure is the 1,3-dialkylimidazolium cation. Its physical and chemical properties can be finely tuned by modifying the alkyl chain lengths (R, R') and selecting appropriate anions (X⁻). For instance, longer alkyl chains can increase lipophilicity, which enhances membrane interactions but may also elevate toxicity. Common anions include [BF₄]⁻, [PF₆]⁻, [Tf₂N]⁻, and halides like Cl⁻ and Br⁻ [15] [23].
• Pharmaceutical Applications: Their primary role has been as green solvents and catalysts in drug synthesis. They contribute to faster reaction rates, higher yields, and easier product separation and solvent recycling compared to conventional volatile organic solvents. A seminal example is the synthesis of the non-steroidal anti-inflammatory drug (NSAID) Pravadoline in [C₄C₁im][PF₆], which achieved a 95% yield and allowed for IL recycling [23]. Beyond synthesis, their tunable hydrophobicity makes them effective for drug solubilization and as components in drug delivery systems to enhance penetration across biological barriers [15] [24].

Cholinium-Based ILs

Driven by the need for biocompatibility, cholinium-based ILs (ChILs) utilize choline, an essential B-group vitamin (Vitamin B4), as the cation. This foundational choice results in ILs with low toxicity and excellent biodegradability profiles.

• Structure and Properties: Choline hydroxide or chloride serves as a common starting material. When combined with biologically relevant anions like amino acids ([AA]⁻), fatty acids (e.g., geranate), or other natural acids, they form a class of ILs known as Cholinium-Amino Acid ILs (ChAAILs) or deep eutectic solvents (DES) [22]. These are often classified as protic ionic liquids (PILs). Their key properties, such as density, viscosity, and conductivity, are significantly influenced by the hydrogen-bonding network spawned by the protic nature and the structure of the anion.
• Pharmaceutical Applications: ChILs are particularly favored in drug delivery and for stabilizing biologics. A prominent example is Choline-Geranate (CAGE), which has been extensively studied for topical and transdermal delivery, enhancing the penetration of various drugs and even showing efficacy in clinical trials for conditions like rosacea and onychomycosis [24]. Their inherent metabolic nature makes them ideal for stabilizing proteins like insulin and for formulating nucleic acid delivery systems, improving stability and cellular uptake without disrupting epithelial integrity [22] [24].

Active Pharmaceutical Ingredient Ionic Liquids (API-ILs)

API-ILs represent a groundbreaking strategy that transcends the role of ILs as mere excipients. In this approach, a therapeutic agent is incorporated as either the cation or the anion of the IL, thereby creating a "liquid drug."

• Concept and Synthesis: The core idea is to convert a solid, crystalline API into a liquid form by pairing it with a suitably selected counterion. This strategy directly addresses common pharmaceutical problems like polymorphism, low aqueous solubility, and limited bioavailability [25] [23]. For example, a common API-IL is formed by pairing the cation 1-alkyl-3-methylimidazolium ([CₙC₁im]⁺) with the anionic form of ibuprofen ([Ibu]⁻) [25].
• Key Advantages:
  • Elimination of Polymorphism: The liquid state inherently avoids crystalline polymorphism, ensuring consistent product quality and performance [23].
  • Enhanced Solubility and Bioavailability: API-ILs can dramatically increase water solubility. For instance, [C₂OHmim][Ibu] exhibits a solubility 10⁵ times higher than crystalline ibuprofen, directly leading to improved bioavailability [25].
  • Dual-Function Therapeutics: It is possible to create API-ILs where both ions possess biological activity, enabling combination therapy in a single ionic entity [15] [23].

Table 1: Comparative Analysis of Major IL Classes in Pharmaceuticals

Feature	Imidazolium-Based ILs	Cholinium-Based ILs	API-ILs
Core Structure	1,3-Dialkylimidazolium cation [15]	Cholinium (Vitamin B4) cation [22]	API as either cation or anion [25]
Key Properties	Thermally stable, tunable hydrophobicity, good solvents [15]	Low toxicity, biodegradable, protic nature [22]	Liquid API, tunable solubility, no polymorphism [23]
Primary Pharma Applications	Drug synthesis (solvent/catalyst), solubilization [15] [23]	Drug delivery (e.g., CAGE), stabilizer for biologics [24]	Liquid formulations, enhanced bioavailability [25]
Toxicity & Biocompatibility	Varies; can be high for early generations [15]	Generally high biocompatibility [22] [24]	Dependent on selected counterion; designed for safety [23]
Example	[C₄C₁im][PF₆] for Pravadoline synthesis [23]	[Ch][Ger] (CAGE) for transdermal delivery [24]	[C₂OHmim][Ibu] for enhanced solubility [25]

Experimental Protocols and Methodologies

Protocol: Synthesis of Cholinium-Amino Acid ILs (ChAAILs)

ChAAILs are typically synthesized via a straightforward acid-base neutralization reaction [22].

• Materials:
  • Choline hydroxide solution ([Ch][OH]) or Choline chloride ([Ch][Cl]) and Potassium hydroxide (KOH).
  • Desired Amino Acid (e.g., Glycine, Alanine, Proline).
  • Solvents: Deionized water, absolute ethanol.
• Procedure (Using [Ch][OH]):
  • Dissolve the chosen amino acid (e.g., 1.0 mol) in a minimal amount of deionized water in a round-bottom flask.
  • Cool the solution in an ice bath to maintain a temperature between 0-5°C.
  • Slowly add an equimolar aqueous solution of choline hydroxide ([Ch][OH]) to the amino acid solution with continuous stirring.
  • Remove the water solvent by rotary evaporation under reduced pressure at 50-60°C.
  • Further dry the resulting viscous liquid in a vacuum desiccator over P₂O₅ for at least 24-48 hours to obtain the pure, anhydrous ChAAIL.
• Procedure (Alternative Metathesis Route):
  • This method uses cheaper [Ch][Cl]. First, prepare the potassium salt of the amino acid by reacting it with KOH in ethanol.
  • Then, react this potassium AA salt with [Ch][Cl] in ethanol. The KCl byproduct precipitates out of the ethanol solution.
  • Filter the mixture to remove the KCl precipitate.
  • Remove ethanol from the filtrate by rotary evaporation and dry the product thoroughly under high vacuum [22].
• Characterization: The resulting IL should be characterized by (^1)H NMR to confirm structure and identity. Karl Fischer titration should be used to determine and ensure low water content.

Protocol: Esterification in Imidazolium ILs as Solvent/Catalyst

This protocol outlines the use of imidazolium ILs as a dual solvent and catalyst, using the esterification of curcumin as a model reaction [15].

• Materials:
  • Ionic Liquid: e.g., 1-butyl-3-methylimidazolium bis(trifluoromethylsulfonyl)imide ([C₄C₁im][N(Tf)₂]).
  • Substrates: Curcumin, Acetic anhydride.
  • Extraction Solvent: Ethyl acetate.
• Procedure:
  • Combine curcumin and a molar excess of acetic anhydride in the selected imidazolium IL (e.g., [C₄C₁im][N(Tf)₂]) without any additional solvent.
  • Stir the reaction mixture at a moderate temperature (e.g., 60-80°C) for a short period (e.g., 15 minutes).
  • Monitor the reaction by TLC or HPLC until completion.
  • Upon completion, cool the mixture. The product, curcumin diacetate, may precipitate or can be extracted.
• Work-up and IL Recycling:
  • Product Isolation: For hydrophilic ILs, add ethyl acetate and water to extract the product into the organic phase. Separate the IL-containing aqueous phase.
  • IL Recovery: Wash the recovered IL phase with fresh ethyl acetate several times to remove any residual organic compounds.
  • Dry the purified IL in a vacuum oven at 50°C for 24 hours before reusing it in subsequent reaction cycles. Studies show ILs like [C₆C₁im][N(Tf)₂] can be recycled at least three times without a significant loss in catalytic activity [15].

Protocol: Investigating Nanostructuring in API-ILs via EPR Spectroscopy

Understanding the molecular-level structure and dynamics of API-ILs is crucial for their application. Electron Paramagnetic Resonance (EPR) spectroscopy with spin probes is a powerful method to study this [25].

• Materials:
  • API-IL of interest (e.g., [Cₙmim][Ibu]).
  • Spin Probes: Stable radicals like TEMPO-D18 or spirocyclohexane-substituted nitroxide (N1).
• Sample Preparation:
  • Dissolve a trace amount (typically < 1 mM) of the spin probe in the API-IL.
  • Ensure homogeneous mixing, potentially by gentle heating and vortexing if the IL is viscous.
  • Transfer the sample to a standard EPR tube.
• EPR Measurements:
  • Use a Continuous Wave (CW) EPR spectrometer to acquire initial spectra.
  • For dynamic information, perform Pulse EPR experiments. Specifically, measure the transverse relaxation time (T₂) at two different spectral positions (I and II) of the echo-detected spectrum using a two-pulse (π/2-τ-π-τ-echo) sequence.
  • Conduct these measurements across a temperature range, typically from low temperature (e.g., 50 K) through the glass transition (T_g) to the liquid state.
• Data Analysis:
  • Calculate the librational parameter, L, from the T₂ values: L = 1/T₂(II) - 1/T₂(I).
  • Plot L as a function of temperature (L(T) dependence).
  • Analyze the plot for regions of linear growth (indicative of stochastic molecular librations) and anomalous regions where librations are suppressed, which signify structural rearrangements within the API-IL glassy matrix. This reveals the nanoscale heterogeneity and structural dynamics critical for the API-IL's stability and performance [25].

The following workflow visualizes the key experimental steps in creating and characterizing a protic ChAAIL and subsequently using EPR to probe its nanostructure.

The Scientist's Toolkit: Essential Research Reagents and Materials

Table 2: Key Reagents and Materials for IL Research in Pharmaceuticals

Reagent/Material	Function/Application	Specific Examples
Imidazolium Salts	Versatile solvents and catalysts in drug synthesis; backbone for creating API-ILs [15] [25].	1-butyl-3-methylimidazolium hexafluorophosphate ([C₄C₁im][PF₆]), 1-hexyl-3-methylimidazolium ibuprofenate ([C₆mim][Ibu]) [25] [23].
Choline Salts	Starting material for synthesizing biocompatible ILs for drug delivery [22] [24].	Choline chloride ([Ch][Cl]), Choline hydroxide solution ([Ch][OH]) [22].
Amino Acids	Anion components for creating low-toxicity, biodegradable ChAAILs [22].	Glycine, Alanine, Proline, Serine [22].
Bio-Acids	Anions for forming biocompatible ILs with specific functional properties [24].	Geranic acid (forms CAGE), Salicylic acid, Fatty acids [24].
Spin Probes	Molecular reporters for studying nanostructuring and dynamics in ILs via EPR spectroscopy [25].	TEMPO-D18 (for CW-EPR), spirocyclohexane-substituted nitroxide N1 (for pulse EPR) [25].
Standard APIs	Model compounds for solubility studies or for conversion into API-ILs to enhance properties [25] [23].	Ibuprofen (acidic API), Barbiturates (acidic API), various amine-containing drugs (basic APIs) [25] [23].

The strategic application of imidazolium, cholinium, and API-IL classes is revolutionizing pharmaceutical research by leveraging the fundamental principle of tunability. Imidazolium ILs provide a powerful toolbox for synthetic chemistry, cholinium ILs offer a biocompatible gateway for advanced delivery and stabilization, and API-ILs fundamentally redefine drug formulation by overcoming the inherent limitations of solid-state actives. As research progresses, the integration of these classes with innovative technologies like AI-driven design and 3D-printing is poised to further expand their potential. The future of ILs in pharma lies in the rational design of smart, multifunctional, and precisely targeted ionic systems that will drive advancements in green chemistry, personalized medicine, and therapeutic efficacy.

From Concept to Formulation: Methodologies and Emerging Applications in Drug Delivery

The development of efficient and safe drug delivery systems represents a paramount objective in modern pharmaceutical research and therapeutic innovation. Conventional delivery platforms face persistent challenges that substantially limit their clinical utility, including poor aqueous solubility of numerous marketed drugs or pipeline candidates (BCS Class II/IV), structural instability under physiological conditions leading to premature drug breakdown, and nonspecific biodistribution resulting in insufficient drug accumulation at target sites while inducing off-target toxicity [24]. These limitations underscore the urgent need for advanced delivery technologies capable of overcoming multiple pharmacological barriers simultaneously.

The convergence of materials science and biomedical engineering has propelled ionic liquids (ILs) to the forefront of next-generation drug delivery solutions. As organic salts that remain liquid below 100°C, ILs exhibit unparalleled molecular design flexibility owing to their modular cation-anion combinations [24] [7]. This structural tunability enables precise tuning of critical pharmaceutical parameters including solubility, stability, and biocompatibility. The programmable nature and multifunctional capabilities of ILs establish a versatile technological platform for precise spatiotemporal regulation of drug delivery, moving beyond conventional systems limited by poor targeting and uncontrolled release [24]. This in-depth technical guide explores the advanced drug loading strategies—ionic/covalent bonding, physical mixing, and nanocarrier encapsulation—that leverage the unique properties of ILs to address longstanding challenges in pharmaceutical development.

Ionic Liquids: Fundamental Properties and Pharmaceutical Classification

Ionic liquids are molten organic salts composed of asymmetrical organic cations paired with inorganic or organic anions, characterized by a melting point ≤ 100°C and extremely low vapor pressure [7]. Their significant versatility stems from easily adjustable chemical properties by altering the structures of their cations and anions. ILs can possess a broad spectrum of polarities, ranging from highly polar to non-polar, depending on the chosen cation and anion combination. The disparity in ion sizes leads to decreased crystallinity in the system, enabling ILs to remain liquid at lower temperatures [7].

Generations and Classes of Pharmaceutical ILs

ILs have evolved through three generations, each suited for different applications based on chemical structure and properties:

• First-generation ILs possess low melting points, high thermal stability, low vapor pressure, and broad fluidity ranges but are sensitive to water and air, non-biodegradable, and exhibit aquatic toxicity [7].
• Second-generation ILs are stable in both air and water with adjustable physical and chemical properties but are characterized by high toxicity and poor biodegradability [7].
• Third-generation ILs include biologically active ions specifically suited for biopharmaceutical applications. These offer low toxicity, reduced manufacturing costs, and good biodegradability compared to earlier generations [7].

Notably, different classes of ILs offer distinctive functional advantages for pharmaceutical applications. Imidazolium-based ILs provide broad thermodynamic stability and structural adaptability, allowing fine-tuning of hydrophobicity and interfacial behavior for enhanced drug solubilization and membrane interactions [24]. Choline-based ILs, derived from an essential nutrient, offer exceptional biocompatibility and are particularly effective for stabilizing biologics and enhancing mucosal permeability without disrupting epithelial integrity [24]. The differentiation between protic and aprotic ILs allows strategic selection based on hydrogen-bonding capability and stability profiles, with protic ILs offering superior solvation capacity and aprotic ILs excelling in formulation stability [24]. Most innovatively, active pharmaceutical ingredient ionic liquids (API-ILs) convert drug molecules directly into ionic forms, markedly improving solubility and bioavailability while integrating the active agent and delivery vector into a single ionic entity [24].

Table 1: Classification of Ionic Liquids for Pharmaceutical Applications

IL Category	Key Components	Pharmaceutical Advantages	Typical Applications
API-ILs	Drug ions as cations/anions	Enhanced bioavailability, eliminates polymorphism	Oral delivery, transdermal systems
Bio-ILs	Cholinium, betainium, amino acids	Low toxicity, good biodegradability	Biologics delivery, topical formulations
SAILs	Long alkyl chains in cation/anion	Self-assembly, micelle formation	Nanocarriers, solubility enhancement
Imidazolium-based	Imidazolium cations	Tunable hydrophobicity, membrane penetration	Permeation enhancement, extraction
Choline-based	Choline cations	High biocompatibility, mucosal penetration	Vaccine adjuvants, protein stabilization

Advanced Drug Loading Strategies: Mechanisms and Methodologies

The unique properties of ionic liquids (ILs) originate from their distinctive molecular architecture and the complex interactions between constituent ions, which collectively determine drug loading behavior. These characteristics are governed by a delicate balance of intermolecular forces, including ionic bonds, hydrogen bonds, van der Waals interactions, as well as potential π-π or n-π stacking effects [24]. Through careful selection of ion pairs and structural modifications, ILs can be engineered with precise drug loading capacities and release profiles. The following sections detail the primary drug loading strategies employed in IL-based delivery systems.

Ionic and Covalent Bonding Strategies

Active Pharmaceutical Ingredient Ionic Liquids (API-ILs)

API-ILs represent the most direct drug loading approach, where the active pharmaceutical ingredient is incorporated as either the cation or anion component of the IL structure. This strategy was first introduced in 2007 with the synthesis of ranitidine docusate [7]. API-ILs are a fascinating area of research due to their potential to enhance solubility, thermal stability, and bioavailability of drugs while addressing issues of polymorphism commonly associated with solid dosage forms [7].

Three types of API-ILs have been identified based on their formation mechanisms [7]:

• Direct Ionic Binding: Created directly through ionic binding using APIs as either anions or cations. This is the most prevalent type where ionizable drugs are paired with biocompatible counterions.

• Covalent Linkage Approach: Involves covalent linkage, forming ionic prodrugs of neutral APIs, which must be synthesized via covalent bonds before being converted into ILs. For instance, neutral paracetamol was transformed into an ionizable form by pairing it with the docusate counterion, resulting in the corresponding IL [7].

• Dual Active API-ILs: Utilizes both methods to produce dual active API-ILs containing two therapeutic agents [7].

Experimental Protocol: Synthesis of API-ILs via Metathesis Reaction

• Pre-ion formation: Dissolve the pharmaceutically active ion (e.g., drug anion) and desired counterion (e.g., choline chloride) in separate containers of deionized water or volatile organic solvent (methanol, acetone).

• Ion exchange reaction: Mix the two solutions with continuous stirring (300-500 rpm) at room temperature for 4-6 hours. For acid-base reactions, directly mix ionic liquid precursor with API.

• Purification: Remove solvent and impurities through rotary evaporation (40-60°C, reduced pressure) or liquid-liquid extraction.

• Drying: Dry the resulting API-IL under high vacuum (0.1-1 mbar) for 12-24 hours at moderate temperatures (50-70°C) to remove residual solvents.

• Characterization: Confirm structure via NMR spectroscopy, assess purity by HPLC, and determine thermal properties by DSC/TGA.

Covalent Drug Conjugation

Beyond simple ionic pairing, drugs can be covalently conjugated to IL ions to create prodrug systems with tailored release kinetics. This approach is particularly valuable for controlling drug release profiles and enhancing targeting specificity.

Experimental Protocol: Covalent Conjugation to IL Scaffolds

• Functional group activation: Activate drug functional groups (carboxyl, hydroxyl, amine) using appropriate activating agents (NHS, EDC, carbonyldiimidazole) in anhydrous DMSO or DMF.

• Coupling reaction: Add functionalized IL (amine-terminated or carboxyl-terminated) to the activated drug solution at molar ratios optimized for the specific chemistry (typically 1:1 to 1:2 drug:IL).

• Reaction monitoring: Stir reaction mixture at room temperature or mild heating (25-40°C) for 12-48 hours, monitoring completion by TLC or HPLC.

• Purification: Purify conjugated product using precipitation in diethyl ether/ethyl acetate, followed by column chromatography if necessary.

• Characterization: Confirm conjugation success via MS (MALDI-TOF or ESI) and NMR spectroscopy, assess drug content by UV-Vis spectrometry.

Physical Mixing and Solubilization

Physical mixing represents a simpler approach where drugs are dissolved or dispersed within ILs without chemical modification. This strategy leverages the exceptional solvation power of ILs to enhance drug loading capacity and stability.

The solvation mechanism involves the disruption of the crystalline lattice of drug molecules by IL ions through various intermolecular interactions including hydrogen bonding, π-π stacking, and van der Waals forces [24]. The extent of solvation is governed by the hydrogen bond basicity and acidity of the IL components, which can be tuned through appropriate selection of cations and anions.

Experimental Protocol: Physical Drug Loading in ILs

• Solubility screening: Conduct preliminary solubility studies by adding excess drug (5-10 mg) to small volumes of IL (1 mL) in microcentrifuge tubes.

• Equilibration: Vortex mixtures thoroughly and incubate at controlled temperatures (25°C, 37°C) with continuous shaking (200 rpm) for 24-48 hours.

• Saturation determination: Centrifuge samples at 10,000-15,000 rpm for 10-15 minutes to separate undissolved drug.

• Quantification: Analyze supernatant drug concentration by HPLC or UV-Vis spectroscopy after appropriate dilution with compatible solvents.

• Stability assessment: Monitor physical and chemical stability of drug-IL mixtures over time (0, 1, 2, 4 weeks) under different storage conditions (4°C, 25°C).

For drugs with particularly low solubility, the following enhancement strategies can be employed:

• Heating-cooling cycles: Heat drug-IL mixtures to 60-80°C for 1-2 hours, then cool slowly to room temperature.
• Lyophilization: Dissolve drug in minimal organic solvent, mix with IL, remove solvent under vacuum, and lyophilize.
• Grinding: Mechanically grind drug and IL together using mortar and pestle or ball milling.

Nanocarrier Encapsulation

ILs can be incorporated into nanocarrier systems to combine the advantages of IL chemistry with the targeting and controlled release capabilities of nanoparticle platforms. This approach includes IL-loaded nanoparticles, IL-stabilized nanocarriers, and IL-involved microemulsions.

Oil-in-ionic liquid nanoemulsions have shown particular promise for vaccine delivery, enhancing both the stability and immune responses of antigens [24] [26]. Similarly, lipid nanoparticles coated with ILs have demonstrated improved siRNA uptake into central nervous system targets [24].

Experimental Protocol: Preparation of IL-Loaded Nanocarriers

• Nanoprecipitation Method:

  • Dissolve polymer (PLGA, PLA) and drug in water-miscible organic solvent (acetone, acetonitrile).
  • Prepare IL-containing aqueous solution with stabilizer (PVA, poloxamer).
  • Rapidly inject organic solution into aqueous phase under high-shear mixing (1000-3000 rpm).
  • Stir continuously for 4-6 hours to evaporate organic solvent.
  • Purify nanoparticles by centrifugation/filtration.

• Emulsion-solvent Evaporation Method:

  • Dissolve polymer and drug in water-immiscible solvent (dichloromethane, ethyl acetate).
  • Emulsify with IL-containing aqueous solution using probe sonication (50-100 W, 2-5 minutes) or high-pressure homogenization (10,000-15,000 psi, 3-5 cycles).
  • Stir emulsion overnight to evaporate organic solvent.
  • Collect nanoparticles by ultracentrifugation.

• Melt Emulsification Method:

  • Heat lipid phase (triglycerides, fatty acids) above melting point (60-80°C).
  • Add IL to aqueous surfactant solution at same temperature.
  • Mix phases using high-speed homogenization to form primary emulsion.
  • Cool to room temperature with continuous stirring to solidify nanoparticles.

Table 2: Characterization Parameters for IL-Based Drug Loading Systems

Characterization Technique	Parameters Assessed	Optimal Specifications
Dynamic Light Scattering	Hydrodynamic diameter, PDI	Size: 50-200 nm, PDI < 0.3
Zeta Potential Analysis	Surface charge		+30	mV for stability
Transmission Electron Microscopy	Morphology, nanostructure	Spherical, uniform shape
High-Performance Liquid Chromatography	Drug loading, encapsulation efficiency	DL > 5%, EE > 80%
Differential Scanning Calorimetry	Thermal behavior, crystallinity	Absence of drug crystals
FTIR Spectroscopy	Chemical interactions, stability	No undesirable interactions
In Vitro Release Study	Drug release profile	Sustained release over 12-72h
Stability Studies	Physical/chemical stability	>90% drug remaining at 4 weeks

The Scientist's Toolkit: Essential Research Reagents and Materials

Successful implementation of IL-based drug loading strategies requires specific reagents and materials optimized for pharmaceutical development. The following table details essential components for research in this field.

Table 3: Essential Research Reagents for IL-Based Drug Formulation

Reagent Category	Specific Examples	Function/Application
IL Cations	Imidazolium, cholinium, pyrrolidinium, ammonium	Structural backbone, property determination
IL Anions	Docusate, acetate, chloride, amino acids	Solubility modulation, biocompatibility
Pharmaceutical Polymers	PLGA, PLA, Chitosan, Alginate	Nanocarrier matrix, controlled release
Surfactants/Stabilizers	Poloxamers, PVA, Tween 80, Span 80	Emulsion stabilization, nanoparticle formation
Biocompatible ILs	Choline geranate (CAGE), Choline lactate	Low toxicity applications, topical delivery
Crosslinkers	Glutaraldehyde, genipin, EDC/NHS	Hydrogel formation, covalent conjugation
Permeation Enhancers	DABCO, azones, bile salts	Barrier penetration, mucosal delivery
Cryoprotectants	Trehalose, mannitol, sucrose	Lyophilization stability, biologics preservation
Analytical Standards	Deuterated solvents, purity standards	Characterization, quantification

Experimental Workflows and Technical Protocols

This section provides detailed methodologies for key experiments cited in recent literature, enabling researchers to replicate and build upon current advances in IL-based drug loading.

Protocol: Transdermal Delivery Formulation with CAGE

Choline geranate (CAGE) has demonstrated remarkable efficacy in enhancing transdermal delivery of both small molecules and macromolecules [24].

Materials:

• Choline bicarbonate and geranic acid (molar ratio 1:2)
• Model drug (e.g., fn14 siRNA for psoriasis treatment)
• Franz diffusion cells
• Excised skin (porcine or human)
• HPLC system for quantification

Method:

• IL Synthesis: Mix choline bicarbonate with geranic acid at 1:2 molar ratio under nitrogen atmosphere. Stir at 60°C for 24 hours. Remove water byproducts under vacuum (0.1 mbar) at 50°C for 6 hours. Confirm structure by 1H NMR [24].

• Drug Loading: Dissolve drug in CAGE at desired concentration (typically 1-5% w/w) by vortexing and gentle heating (37°C) if necessary.

• Skin Permeation Studies:

  • Mount excised skin between donor and receptor compartments of Franz diffusion cells.
  • Apply 200-500 μL drug-loaded CAGE to donor chamber.
  • Maintain receptor phase (PBS with preservatives) at 37°C with continuous stirring.
  • Sample receptor fluid at predetermined intervals (0, 2, 4, 8, 12, 24, 48h) and replace with fresh medium.
  • Analyze samples by HPLC or other appropriate analytical methods.

• Data Analysis: Calculate cumulative drug permeation, flux, and permeability coefficients. Compare with conventional formulations.

Protocol: API-IL Synthesis and Characterization

Materials:

• Ionizable drug (acidic or basic)
• Appropriate counterion source (e.g., choline chloride for acidic drugs)
• Organic solvents (methanol, acetone, dichloromethane)
• Rotary evaporator
• High-vacuum pump

Method:

• Ion Exchange Reaction:
  • Dissolve drug (2 mmol) in minimum amount of methanol.
  • Dissolve counterion source (2 mmol) in separate container.
  • Mix solutions and stir at room temperature for 6-12 hours.

• Purification:

  • Remove solvent under reduced pressure using rotary evaporator.
  • Wash residue with ethyl acetate or diethyl ether to remove unreacted starting materials.
  • Dry under high vacuum (0.1 mbar) for 24 hours.

• Characterization:

  • NMR Spectroscopy: Confirm chemical structure and purity.
  • DSC/TGA: Determine melting point, thermal stability, and decomposition profile.
  • Solubility Studies: Measure solubility in water and biorelevant media compared to parent drug.
  • In Vitro Release: Assess drug release in simulated gastric and intestinal fluids.

Protocol: IL-Stabilized Nanocarrier Preparation

Materials:

• Biodegradable polymer (PLGA, PLA)
• Ionic liquid (imidazolium or choline-based)
• Surfactant (PVA, poloxamer 188)
• Organic solvent (ethyl acetate, dichloromethane)
• Probe sonicator or high-pressure homogenizer

Method:

• Organic Phase Preparation: Dissolve polymer (100 mg) and drug (5-20 mg) in organic solvent (5 mL).

• Aqueous Phase Preparation: Dissolve IL (0.1-1% w/v) and surfactant (1-2% w/v) in aqueous medium (20 mL).

• Emulsification:

  • Add organic phase to aqueous phase dropwise with probe sonication (50-100 W, 2-5 minutes) or high-pressure homogenization (15,000 psi, 3 cycles).
  • Stir emulsion overnight to evaporate organic solvent.

• Purification and Characterization:

  • Centrifuge at 15,000 rpm for 30 minutes and wash particles.
  • Resuspend in cryoprotectant solution (5% trehalose) for lyophilization.
  • Characterize particle size, zeta potential, drug loading, and in vitro release profile.

Visualization of Drug Loading Mechanisms and Workflows

The following diagrams illustrate key concepts, relationships, and experimental workflows in IL-based drug loading strategies.

IL Drug Loading Mechanisms

Diagram 1: IL Drug Loading Strategies and Mechanisms

API-IL Synthesis Workflow

Diagram 2: API-IL Synthesis and Characterization Workflow

Ionic liquids have redefined drug delivery paradigms through molecular-level programmability, enabling breakthrough strategies such as ionic/covalent drug conjugation, nanoencapsulation, and stimuli-responsive carrier design. These innovations overcome persistent limitations of conventional systems, including poor solubility of hydrophobic drugs, instability of biologics, and inadequate penetration across physiological barriers [24]. Notable successes include the dramatic enhancement of transdermal delivery using choline-geranic acid ILs (CAGE), stabilization of vaccine antigens through oil-in-IL nanoemulsions, and significant improvement in oral bioavailability of BCS Class II/IV drugs [24] [26].

Despite these promising advances, challenges remain in the clinical translation of IL-based drug delivery systems. High production costs, regulatory barriers, and the need for comprehensive long-term toxicity studies present hurdles for widespread adoption [7] [27]. However, ongoing research and federal funding are driving innovation, making ILs more economically viable. Future development should focus on rational design of biocompatible ILs from pharmaceutical-grade ions, comprehensive safety profiling, industrial scale-up of manufacturing processes, and clinical validation of efficacy [24] [7].

The integration of emerging technologies like AI-driven design and 3D-printed IL formulations promises to accelerate the development of intelligent, precision IL-based delivery systems poised for clinical adoption [24]. As research continues to address current limitations and expand applications, ionic liquids are positioned to become a cornerstone of advanced drug delivery, offering sustainable, efficient, and tunable platforms for next-generation therapeutics.

Ionic liquid self-assembled micelles (ILs-SAMs) represent a advanced class of drug delivery systems that synergistically combine the unique properties of ionic liquids (ILs) with the structural benefits of micellar nanocarriers [28] [29]. As organic salts with melting points below 100°C, ILs possess unparalleled molecular design flexibility owing to their modular cation-anion combinations [24]. This structural tunability enables precise control of critical pharmaceutical parameters including solubility, stability, and biocompatibility [24]. The convergence of ILs with micelle technology has created multifunctional platforms capable of overcoming persistent drug delivery challenges, including poor bioavailability of hydrophobic drugs, structural instability under physiological conditions, and nonspecific biodistribution [24]. ILs-SAMs are micellar structures formed by the self-assembly of surface-active ionic liquids (SAILs) through various intermolecular interactions, affected by multiple environmental factors [29]. These systems possess unique physicochemical properties that confer high stability and effective drug carrier capabilities, with compositions and structures that can be regulated for flexible and controllable characteristics [29]. This technical guide comprehensively examines the assembly mechanisms, characterization methodologies, functional properties, and therapeutic applications of ILs-SAMs, providing researchers with essential knowledge for exploiting these versatile nanocarriers in pharmaceutical development.

Fundamental Principles and Assembly Mechanisms

Molecular Architecture of Ionic Liquids

The self-assembly capability of ILs into micellar structures originates from their distinctive molecular architecture, characterized by a delicate balance of intermolecular forces including ionic bonds, hydrogen bonds, van der Waals interactions, and potential π-π or n-π stacking effects [24]. ILs suitable for micelle formation typically consist of amphiphilic molecules containing both hydrophobic and hydrophilic regions [30]. The structural diversity of ILs encompasses several key categories:

• Imidazolium-based ILs: Provide broad thermodynamic stability and structural adaptability, allowing fine-tuning of hydrophobicity and interfacial behavior for enhanced drug solubilization and membrane interactions [24].
• Choline-based ILs: Derived from an essential nutrient, these offer exceptional biocompatibility and are particularly effective for stabilizing biologics and enhancing mucosal permeability without disrupting epithelial integrity [24].
• Protic versus aprotic ILs: This differentiation allows strategic selection based on hydrogen-bonding capability and stability profiles, with protic ILs offering superior solvation capacity and aprotic ILs excelling in formulation stability [24].
• Active Pharmaceutical Ingredient Ionic Liquids (API-ILs): Represent the most innovative approach, converting drug molecules directly into ionic forms to markedly improve solubility and bioavailability while integrating the active agent and delivery vector into a single ionic entity [24].

Assembly Mechanisms and Driving Forces

The self-assembly of ILs into micellar structures is governed by complex interplay between various molecular forces and environmental parameters. The primary driving force is the hydrophobic effect, where non-polar segments aggregate to minimize contact with aqueous environments, while ionic head groups remain hydrated at the micelle-water interface [29]. This process results in the formation of nanoscale structures with typically core-shell architectures.

Table 1: Critical Parameters Influencing ILs-SAMs Assembly

Parameter Category	Specific Factors	Impact on Assembly
Molecular Structure	Alkyl chain length, Head group chemistry, Counterion type	Determines CMC values, micelle size, morphology, and stability
Environmental Conditions	Temperature, pH, Ionic strength	Affects aggregation behavior, micelle size, and drug loading capacity
Solution Properties	Concentration, Solvent composition	Influences micellization kinetics and final nanostructure

The critical micelle concentration (CMC) represents a key parameter defining the minimum IL concentration required for spontaneous micelle formation [30]. CMC values are influenced by the structural features of ILs, with longer alkyl chains typically lowering the CMC due to enhanced hydrophobic interactions [30]. Specific molecular modifications, such as additional methyl groups in the backbone of the imidazolium core, can significantly impact molecular interactions by altering the hydrogen-bonding network [31].

Figure 1: ILs-SAMs Self-Assembly Pathway. The diagram illustrates the sequential process from discrete IL monomers to functional drug-loaded micelles, highlighting the critical role of concentration-driven aggregation.

Beyond conventional spherical micelles, ILs can form more complex architectures including cubosomes - micro- and nanoparticles with a bicontinuous cubic two-phase structure characterized by maximum continuous interface and high interface-to-volume ratio [32]. These structures are particularly promising for efficient adsorbents and host-guest applications due to their unique structural properties [32].

Characterization Techniques and Methodologies

Essential Characterization Methods

Comprehensive characterization of ILs-SAMs requires multidisciplinary approaches to elucidate their structural, morphological, and functional properties. Key techniques employed in current research include:

• Dynamic Light Scattering (DLS) and Static Light Scattering (SLS): Provide hydrodynamic diameter, size distribution, and aggregation number of micelles in solution [28]. The polydispersity index (PDI) derived from DLS measurements indicates the homogeneity of micelle populations [28].
• Transmission Electron Microscopy (TEM) and Cryo-TEM: Enable direct visualization of micelle morphology, size, and internal structure [28] [33]. Cryo-TEM preserves the native state of micelles in aqueous environments without drying artifacts [33].
• Small-Angle X-ray Scattering (SAXS) and Small-Angle Neutron Scattering (SANS): Probe nanoscale structure and periodic arrangements within micellar assemblies, providing information about core-shell dimensions and internal ordering [28].
• Surface Tension Measurements: Determine critical micelle concentration (CMC) values by tracking surface tension as a function of IL concentration [30]. The CMC represents the concentration at which a distinct breakpoint appears in the surface tension curve.
• Nuclear Magnetic Resonance (NMR) Spectroscopy: Elucidates molecular interactions between ILs and encapsulated drugs, providing insights into molecular orientation and binding mechanisms [30].
• Fluorescence Spectroscopy: Employed using probes like pyrene to investigate micelle microenvironments and determine CMC values through changes in fluorescence intensity ratios [30].

Table 2: Key Characterization Techniques for ILs-SAMs

Technique	Parameters Measured	Experimental Considerations
Dynamic Light Scattering (DLS)	Hydrodynamic diameter, PDI	Requires appropriate concentration; sensitive to dust
Transmission Electron Microscopy (TEM)	Micelle morphology, size	May require staining; vacuum conditions
Critical Micelle Concentration (CMC)	Aggregation onset concentration	Surface tension vs. concentration plot
Nuclear Magnetic Resonance (NMR)	Molecular interactions, structure	Deuterated solvents; concentration-dependent studies
Fluorescence Spectroscopy	Microenvironment polarity, CMC	Probe selection critical; concentration optimization

Experimental Protocols for ILs-SAMs Characterization

CMC Determination via Surface Tension Method

Materials: IL sample in purified form, double-distilled water, Du Noüy ring tensiometer or equivalent, constant temperature bath.

Procedure:

• Prepare stock solution of IL at concentration above expected CMC.
• Serially dilute to obtain concentration series (typically 10-15 points spanning expected CMC).
• Equilibrate each solution at constant temperature (25°C) for 15 minutes.
• Measure surface tension for each concentration using calibrated tensiometer.
• Plot surface tension versus logarithm of concentration.
• Determine CMC from intersection point of two linear regions pre- and post-micellization.

Validation: Perform measurements in triplicate; confirm with alternative method (fluorescence spectroscopy) for key samples.

Micelle Size and Morphology Analysis

Materials: Purified ILs-SAMs solution, appropriate buffers, DLS instrument, TEM with negative staining capability.

DLS Procedure:

• Filter ILs-SAMs solution through 0.22 μm or 0.45 μm filter to remove dust.
• Adjust concentration to optimal range for instrument (typically 0.1-1 mg/mL).
• Equilibrate at measurement temperature (25°C) for 5 minutes.
• Perform measurements at multiple angles (if multi-angle DLS).
• Analyze correlation function using appropriate algorithms (Cumulants, CONTIN) to obtain size distribution.

TEM Procedure:

• Apply diluted ILs-SAMs solution (5-10 μL) to carbon-coated grid.
• Blot excess solution after 1-2 minutes.
• Negative stain with uranyl acetate (2%) or phosphotungstic acid (1%) for 30 seconds.
• Air dry completely before TEM observation.
• Acquire images at multiple magnifications; measure sizes from multiple particles (n>100).

Functional Properties and Enhancement Mechanisms

Solubilization Enhancement

ILs-SAMs dramatically improve the solubility of poorly water-soluble drugs through multiple mechanisms that operate simultaneously. The core-shell structure of micelles provides distinct hydrophobic and hydrophilic regions that can accommodate drugs of varying polarity [24]. The unique microenvironments within ILs-SAMs differ substantially from bulk water, creating favorable conditions for drug solubilization [24]. Specific molecular interactions, including π-π stacking, ion-dipole interactions, and hydrogen bonding, further enhance drug incorporation beyond simple partitioning [30]. Studies have demonstrated that ILs containing specific structural features, such as phenyl groups in amino acid-based SAILs, exhibit particularly strong interaction capabilities with drug molecules [30].

Permeation Enhancement

ILs-SAMs significantly enhance drug permeation across biological barriers, particularly skin, through multifaceted mechanisms. They alter the surface tension and viscosity of permeating substances, facilitating transdermal drug penetration [29]. IL constituents can interact with stratum corneum lipids, temporarily disrupting their highly ordered structure to create pathways for drug diffusion [24]. The nanoscale size of ILs-SAMs enables efficient follicular penetration and intercellular transport through skin layers [29]. Research has demonstrated remarkable success in transdermal delivery applications, with ILs-SAMs facilitating skin penetration of various therapeutic agents including small molecules, peptides, and even nucleic acids like siRNA [24].

Stabilization and Controlled Release

ILs-SAMs provide exceptional stabilization for labile therapeutic agents, including peptides, proteins, and nucleic acids, against enzymatic degradation and structural denaturation [30]. The confined microenvironments within micelles shield encapsulated molecules from destructive influences while maintaining their biological activity [24]. The programmable nature of ILs enables sophisticated controlled release profiles through stimulus-responsive design strategies [24]. Environmental triggers such as pH changes, enzymatic activity, or temperature variations can be exploited to modulate drug release kinetics at target sites [24].

Figure 2: Multifunctional Mechanisms of ILs-SAMs. The diagram illustrates the four primary functional properties of ILs-SAMs and their underlying structural mechanisms that enable enhanced drug delivery.

Structure-Function Relationships and Biocompatibility

Cationic Alkyl Chain Length Effects

Systematic studies have revealed that the length of the cationic alkyl chain in ILs represents a dominant factor governing both self-assembly behavior and biological compatibility [33]. Research utilizing comprehensive IL libraries has demonstrated a clear correlation between alkyl chain length and cytotoxicity across multiple cell lines, including mouse brain endothelial (bEnd.3) cells, mouse breast cancer (4T1) cells, and human hepatocellular carcinoma (HepG2) cells [33]. ILs with short cationic alkyl chains (scILs, C1-C4) exhibit minimal cytotoxicity, while those with long cationic alkyl chains (lcILs, ≥C8) show dramatically increased toxicity [33]. This structure-activity relationship has been validated in more complex biological systems including 3D cell spheroids and patient-derived organoids, confirming the superior biocompatibility profile of scILs [33].

Nanoaggregate-Cell Interactions

The biological interactions of ILs occur primarily through their nanoaggregate forms rather than as individual molecules [33]. Cryo-TEM studies have visualized these nanoaggregates, with scILs typically forming smaller structures (~5 nm) compared to lcILs (~12.5 nm) [33]. These dimensional differences profoundly influence cellular uptake and intracellular trafficking pathways. scILs are predominantly restricted within intracellular vesicles, while lcILs accumulate in mitochondria, inducing mitophagy and apoptosis [33]. Molecular dynamics simulations confirm that amphiphilicity serves as the driving force for nanoaggregate formation, with cationic alkyl chains embedded inside cationic heads paired with anions in aqueous environments [33].

In Vivo Behavior and Administration Route Considerations

The favorable biocompatibility profile of scILs translates to superior in vivo tolerance across multiple administration routes [33]. Animal studies demonstrate that scILs exhibit approximately 30-80 times greater tolerance than lcILs regardless of administration pathway (oral, intramuscular, or intravenous) [33]. The oral route demonstrates particularly advantageous compliance and tolerance characteristics while enabling enhanced bioavailability of insoluble drugs when formulated with scIL nanoaggregates as carriers [33].

Research Reagent Solutions

Table 3: Essential Research Reagents for ILs-SAMs Investigation

Reagent Category	Specific Examples	Function and Application
SAILs (Surface-Active ILs)	[C₈mim][Gly], [C₂mim][Ole], [Cho][Ole]	Primary micelle formers; backbone of delivery system [30]
Characterization Standards	Polystyrene nanospheres, Latex size standards	Instrument calibration for DLS, TEM [28]
Fluorescence Probes	Pyrene, Nile Red, ANS	CMC determination, microenvironment polarity [30]
Biocompatibility Assays	CCK-8, Live/Dead stains, Annexin V	Cytotoxicity evaluation, cell viability assessment [33]
Stabilizers and Additives	Poloxamers, Polysorbates, Lipids	Micelle stability enhancement, surface modification [29]

Therapeutic Applications

ILs-SAMs have demonstrated significant potential across diverse therapeutic areas, particularly in transdermal drug delivery for treating various skin disorders and systemic conditions [29]. Their multifunctional capabilities enable targeted intervention in numerous disease states:

• Skin Cancer: ILs-SAMs facilitate targeted delivery of chemotherapeutic agents to tumor sites while minimizing systemic exposure [29]. The enhanced permeability and retention (EPR) effect further promotes accumulation in malignant tissues [28].
• Arthritis: ILs-SAMs enable effective transdermal delivery of anti-inflammatory agents for rheumatoid arthritis treatment, with demonstrated efficacy in collagen-induced arthritis models [28] [29].
• Diabetes: Micellar systems incorporating ILs have been developed for transdermal insulin delivery, providing sustained release profiles and improved bioavailability compared to conventional administration [29].
• Fungal Infections: ILs-SAMs enhance skin penetration of antifungal agents like ketoconazole, significantly improving treatment efficacy against dermatophytoses [29].
• Skin Photoaging: Antioxidant-loaded ILs-SAMs protect against oxidative stress and collagen degradation, offering innovative approaches to cosmetic dermatology [29].

ILs-SAMs represent a transformative platform in drug delivery science, combining the exceptional tunability of ionic liquids with the proven benefits of micellar nanocarriers. Their programmable assembly mechanisms, multifunctional capabilities, and demonstrated therapeutic efficacy position them at the forefront of next-generation delivery technologies. The structure-activity relationships elucidated through systematic research, particularly regarding alkyl chain length effects on biocompatibility, provide essential guidance for rational design of safe and effective formulations. As research advances, ILs-SAMs are poised to overcome persistent challenges in pharmaceutical development, enabling precise spatiotemporal control of drug delivery while enhancing therapeutic outcomes across diverse disease states.

Ionic liquids (ILs), a class of salts that are liquid below 100 °C, have emerged as a transformative material in biomedical research due to their highly tunable physicochemical properties [34] [3]. This tunability stems from the modular combination of various cations and anions, allowing scientists to design ILs with precise characteristics for specific applications [35]. The evolution of ILs has progressed through generations, from first-generation solvents to the current third-generation ILs that incorporate bio-derived, task-specific functionalities with enhanced biocompatibility [5]. For transdermal drug delivery, this tunability is particularly valuable for overcoming the significant barrier function of the skin, especially for large, hydrophilic biologics such as insulin and small interfering RNA (siRNA) that traditionally require injection [36] [37].

The ability to fine-tune IL properties allows researchers to engineer solutions for two fundamental challenges in transdermal delivery: enhancing skin permeation and maintaining the stability of therapeutic molecules. By selecting appropriate ion pairs, ILs can be designed to disrupt the skin's stratum corneum reversibly, facilitate drug solubility, and provide a stable environment for fragile biologics [35]. This guide explores the application of these tunable materials as permeation enhancers for biologics, providing technical details on mechanisms, formulations, and experimental methodologies.

Structural Design and Key Properties of Ionic Liquids

Ionic Liquid Composition and Generations

Ionic liquids are composed of asymmetric, bulky organic cations and smaller organic or inorganic anions, which results in poor crystal packing and low melting points [35] [3]. Their development is categorized into distinct generations:

• First Generation: Focused on their utility as green solvents with unique physical properties, but with limited consideration of biological compatibility [3].
• Second Generation: Engineered for specific application needs such as catalysis and electrochemical systems, with improved stability [5] [3].
• Third Generation: Designed with an emphasis on biocompatibility, incorporating bio-inspired cations like cholinium and task-specific functionalities for biomedical and environmental applications [5]. These generations exhibit low toxicity and are often biodegradable, making them suitable for drug delivery [5] [37].

Table 1: Common Cations and Anions Used in Biocompatible Ionic Liquids for Transdermal Delivery

Cation Type	Example	Anion Type	Example	Key Characteristics
Cholinium	Choline	Carboxylate	Geranate, Decanoate, Salcaprozate	Low toxicity, biodegradable, effective permeation enhancement [36] [38] [37]
Imidazolium	1-butyl-3-methylimidazolium	Fluorinated	Hexafluorophosphate (PF₆⁻), Bis(trifluoromethylsulfonyl)imide	Early prominence, but higher toxicity concerns compared to cholinium [34] [35]
Quaternary Ammonium	Tetraalkylammonium	Amino Acid	Amino acid derivatives	Biocompatible, tunable properties [35]

Tunable Physicochemical Properties

The structure-property relationships in ILs enable precise control over their behavior, which is critical for transdermal formulation design:

• Viscosity: Increases with the length of the alkyl chain on the cation due to stronger van der Waals interactions [3]. This property affects diffusion rates and workability.
• Solubility: Can be tailored to dissolve both hydrophilic and hydrophobic compounds, including poorly water-soluble Active Pharmaceutical Ingredients (APIs) [34] [35].
• Thermal Stability: ILs exhibit high thermal stability, often decomposing only at temperatures above 300 °C, which is beneficial for manufacturing and storage [39].
• Ionic Conductivity: Influenced by the choice of anion, with charge distribution and delocalization leading to higher conductivity [3].

The concept of "task-specific" or "designer" solvents is a cornerstone of IL technology, allowing researchers to select cation-anion pairs that optimally balance permeation enhancement, drug stability, and biocompatibility for a given biologic [5] [3].

Mechanisms of Transdermal Enhancement by Ionic Liquids

Ionic liquids enhance skin permeation through multiple, often simultaneous, mechanisms that reversibly compromise the skin's primary barrier, the stratum corneum (SC). The following diagram illustrates the primary mechanisms by which ILs facilitate the transdermal delivery of biologics.

The efficacy of ILs is attributed to their ionic nature and capacity for diverse molecular interactions. They act on the intercellular lipid matrix, which is crucial for the barrier function of the SC, through mechanisms such as lipid extraction and fluidization [35]. Molecular dynamics simulations suggest that IL aggregates can insert into and modulate lipid bilayers, thereby reducing barrier resistance [36]. Furthermore, certain ILs have been shown to interact with keratin in corneocytes and reversibly modulate tight junction proteins in deeper epidermal layers, facilitating both transcellular and paracellular transport routes for macromolecules [37].

Experimental Protocols and Workflows

This section details the core methodologies for formulating IL-based transdermal systems and evaluating their performance, from preparation to in vivo assessment.

Synthesis and Formulation of Ionic Liquids

Protocol 1: Synthesis of Choline-Based Ionic Liquids via Acid-Base Neutralization

This is a common method for preparing biocompatible ILs, such as choline geranate (CAGE) or choline decanoate [38] [40].

• Reaction Setup: Dissolve the organic acid (e.g., geranic acid, decanoic acid) in a dry, low-boiling-point solvent like anhydrous ethanol or methanol. Typical molar ratios of acid to choline are 2:1.
• Neutralization: Slowly add an aqueous solution of choline bicarbonate or choline hydroxide to the acid solution with continuous stirring at room temperature.
• CO₂ Evolution: The reaction will produce CO₂ bubbles. Continue stirring until gas evolution ceases, indicating reaction completion.
• Solvent Removal: Remove the solvent and water first by rotary evaporation and then by freeze-drying. Further dry the resulting viscous liquid under high vacuum (e.g., at 60 °C for 24 hours) to achieve a water content of <1% w/w, which is critical for the stability of the IL and the encapsulated biologic [38].
• Characterization: Confirm successful synthesis and purity using ¹H and ¹³C NMR spectroscopy. Characterize thermal properties (melting point, glass transition) via Differential Scanning Calorimetry (DSC) [38] [40].

Protocol 2: Preparation of IL-siRNA or IL-Peptide Complexes

For nucleic acids or proteins, the formulation process focuses on maintaining stability.

• IL Pre-conditioning: Ensure the synthesized IL is at the desired temperature and viscosity. High-viscosity ILs may require gentle warming to facilitate mixing.
• Complexation: Add the aqueous solution of the biologic (siRNA or insulin) directly to the IL. The ratio of IL to biologic is critical. For siRNA, a 3:1 (IL:siRNA) ratio has been identified as optimal for stability and delivery [40].
• Homogenization: Mix thoroughly but gently using a vortex mixer or low-speed stirring to form a homogeneous formulation without degrading the biologic.
• Stability Assessment: Analyze the structural integrity of the biologic post-formulation. For siRNA, use native gel electrophoresis and Circular Dichroism (CD) spectroscopy to confirm that the secondary structure remains intact [36].

The workflow below illustrates the key stages of developing and testing an IL-based transdermal formulation.

In Vitro and Ex Vivo Permeation Studies

Protocol 3: Skin Permeation Assessment using Franz Diffusion Cells

This is the gold-standard method for evaluating transdermal delivery ex vivo.

• Skin Membrane Preparation: Use full-thickness or dermatomed porcine or human skin. Porcine skin is a widely accepted model due to its similarity to human skin.
• FDC Assembly: Mount the skin membrane between the donor and receptor compartments of the Franz cell. The dermal side should face the receptor chamber, which is filled with a suitable buffer (e.g., PBS, pH 7.4) and maintained at 37°C with continuous stirring to ensure sink conditions.
• Formulation Application: Apply a measured quantity of the IL-biologic formulation (e.g., 100-500 µL) to the stratum corneum in the donor compartment.
• Sampling: At predetermined time intervals, withdraw samples from the receptor compartment and replace with fresh buffer.
• Analysis: Quantify the amount of permeated biologic using appropriate analytical techniques (e.g., HPLC for insulin, fluorescence spectroscopy for labeled siRNA). Calculate cumulative permeation and flux.
• Skin Deposition: At the end of the experiment, wash the skin surface and tape-strip or homogenize the skin to determine the amount of drug retained within the skin layers [36].

In Vivo Efficacy and Safety Assessment

Protocol 4: Evaluating Therapeutic Efficacy in Disease Models

• Animal Model Induction:
  • Psoriasis Model: Apply imiquimod cream on the shaved back skin of mice to induce psoriasis-like lesions [36] [40].
  • Diabetes Model: Induce diabetes in rodents using streptozotocin (STZ) to create an insulin-deficient model [37].
• Formulation Administration: Topically apply the IL-biological formulation (e.g., IL-siRNA for psoriasis, IL-insulin for diabetes) to the treatment area. Include control groups (e.g., untreated, placebo IL, subcutaneous injection).
• Efficacy Endpoints:
  • Psoriasis: Monitor Psoriasis Area and Severity Index (PASI) scores, measure epidermal thickness via histology, and analyze cytokine levels (e.g., TNF-α, IL-17A) in skin tissue [36] [40].
  • Diabetes: Monitor blood glucose levels at regular intervals post-application. Calculate the relative pharmacologic availability compared to subcutaneous injection [38] [37].
• Safety and Biocompatibility:
  • Histopathology: After the study, excise treated skin and major organs for histological examination (H&E staining) to assess irritation, changes in villi morphology, or other toxicological impacts [38].
  • Cytotoxicity Assays: Perform in vitro assays on cell lines to assess cytotoxicity.

Quantitative Data and Performance of IL-Based Formulations

The following tables summarize key experimental results from recent studies, demonstrating the efficacy of ILs in delivering biologics.

Table 2: Performance of IL-siRNA Formulations in Psoriasis Models

IL Formulation	siRNA Target	Key Findings	Reference
CAGE + CAPA (1:1)	NFKBIZ	~0.4 nmol/cm² epidermal delivery of siRNA; Suppression of psoriasis-related genes (TNF-α, IL-17A) in an imiquimod-induced mouse model.	[36]
CIL ([Ch]₂[Ger]₃ + [Ch][Hyd]₂)	Fn14	Effective delivery and Fn14 silencing in keratinocytes; Ameliorated psoriasis lesions by modulating T-cell activation and reducing keratinocyte proliferation in a mouse model.	[40]

Table 3: Performance of IL-Peptide Formulations for Insulin Delivery

IL Formulation	Administration Route	Key Findings	Reference
Choline Decanoate (chC10 1:2)	Oral (in rats)	6.5% oral bioavailability of insulin; 13-fold higher than sodium decanoate (0.5%) and 7-fold higher than CAGE (0.9%).	[38]
Choline-Salcaprozate ([Ch]₂[Sal])	Intranasal & Sublingual (in mice)	Robust hypoglycemic effect; Reversible modulation of intercellular junctions without irreversible damage to the mucosal epithelium.	[37]

The Scientist's Toolkit: Essential Research Reagents and Materials

Table 4: Key Reagents for IL-Based Transdermal Formulation Research

Reagent/Material	Function in Research	Example Sources / Notes
Choline Bicarbonate/Hydroxide	Common, biocompatible cation precursor for IL synthesis.	Commercial suppliers (e.g., Bide Pharmatech). Aqueous solutions (e.g., 45-80% w/w) are typically used.	[37] [40]
Organic Acids (Geranic, Decanoic, Salcaprozate)	Anion precursors that determine IL's permeation enhancement and physicochemical properties.	Energy Chemical Co., Ltd.; Salcaprozate is the free acid form of the permeation enhancer SNAC.	[36] [38] [37]
Franz Diffusion Cells	Standard apparatus for ex vivo assessment of skin permeation kinetics.	Glass-based systems with a defined diffusion area and a thermostated receptor chamber.	[36]
Porcine Ear Skin	A widely accepted ex vivo model for human skin permeation studies due to similar structure and permeability.	Typically sourced from local abattoirs; should be used fresh or stored frozen for short periods.	[36]

Ionic liquids represent a paradigm shift in transdermal and topical delivery, moving from simple solvents to sophisticated, tunable platforms that can be engineered to overcome specific biological barriers. The modular nature of ILs allows researchers to design formulations that not only enhance permeation but also protect the integrity of complex biologics like siRNA and insulin. As research progresses, the focus will increasingly be on refining the biocompatibility and safety profiles of these materials, scaling up synthesis, and navigating the regulatory pathway. The integration of computational design and high-throughput screening will further accelerate the development of next-generation ILs, solidifying their role as a powerful tool in the drug delivery scientist's arsenal.

Ionic liquids (ILs), a class of materials often defined as organic salts with melting points below 100°C, have emerged as a transformative platform in biopharmaceutical development due to their highly tunable nature and unique physicochemical properties [5] [15]. Their evolution is categorized into generations, with later generations (third- and fourth-generation ILs) focusing on sustainability, biodegradability, and multifunctionality for biomedical applications [5]. The core principle that makes ILs particularly valuable for stabilizing sensitive biomolecules is their designer solvent characteristic; their physical, chemical, and biological properties can be precisely tailored by selecting different combinations of organic cations and organic or inorganic anions [15] [24]. This modularity allows scientists to engineer ILs with specific features—such as desired hydrophilicity, viscosity, protein-binding affinity, or enzymatic resistance—to address the distinct instability challenges presented by proteins, peptides, and nucleic acids [24].

The transition of ILs from green solvents in the first generation to functional materials in biomedical fields has unlocked new strategies for overcoming persistent challenges in biopharmaceutical formulation [5]. These challenges include the physical and chemical instability of proteins (such as aggregation, deamidation, and oxidation), the susceptibility of nucleic acids to enzymatic degradation, and the poor solubility and bioavailability of many therapeutic molecules [41] [24]. By leveraging the diverse interactions ILs can form with biomolecules (e.g., electrostatic, hydrogen bonding, and hydrophobic interactions), researchers can create a stable microenvironment that protects these therapeutics from degradation throughout manufacturing, storage, and delivery [15].

Mechanisms of Protein and Peptide Stabilization

Proteins and peptides are inherently complex and prone to a range of degradation pathways, including unfolding, aggregation, deamidation, and oxidation [41] [42]. Ionic liquids can mitigate these pathways through multiple mechanisms that preserve the native structure and function of the biologic.

Suppression of Aggregation and Stabilization of Structure

Protein aggregation is a major concern that can reduce drug efficacy and increase immunogenicity risk [41]. ILs can counteract this through direct interaction with the protein structure:

• Prevention of Unfolding: Certain ILs, particularly choline-based varieties, interact with protein surfaces via hydrogen bonding and ionic interactions. This interaction can stabilize the native conformation, making the protein more resistant to thermal- or stress-induced unfolding, which is a common precursor to aggregation [15] [24].
• Inhibition of Non-covalent Aggregation: By modulating the solvation shell around the protein, ILs can reduce protein-protein interactions that lead to the formation of non-covalent aggregates. Studies have shown that ILs can suppress aggregation during refolding processes and long-term storage [15].
• Modulation of Protein-Protein Interactions: The ions of an IL can localize to the protein surface and alter its charge and hydration properties. This can create an electrosteric barrier that prevents the close approach of protein molecules necessary for aggregation to occur [24].

The stabilization effect is highly dependent on the specific ion combinations. For instance, choline dihydrogen phosphate has been extensively studied for its ability to stabilize the native state of various proteins, including monoclonal antibodies [15].

Protection Against Chemical Degradation

Proteins are susceptible to chemical degradation such as deamidation of asparagine, oxidation of methionine, and hydrolysis of the peptide backbone [42]. ILs can offer protection by creating a stabilizing microenvironment:

• Deamidation and Hydrolysis: These reactions are highly dependent on pH and the presence of water. ILs can control local water activity and provide a stable pH buffer, thereby slowing down these degradation pathways [24] [42].
• Oxidation: The oxidation of susceptible residues (e.g., methionine, cysteine, tryptophan) can be catalyzed by metal ions or light. Some ILs can chelate metal ions or form a protective layer that shields the protein from reactive oxygen species [42].

Table 1: Ionic Liquids and Their Documented Stabilization Effects on Proteins and Peptides

Ionic Liquid (Example)	Target Biomolecule	Stabilization Effect	Postulated Primary Mechanism
Choline Dihydrogen Phosphate	Monoclonal Antibodies, Lysozyme	Reduced aggregation, enhanced thermal stability	Strong hydrogen bonding, preferential exclusion
Imidazolium-based ILs ([C4mim][Cl])	Green Fluorescent Protein (GFP)	Suppressed aggregation, maintained native structure	Modulation of protein-protein interactions
Choline Geranate (CAGE)	Insulin	Enhanced structural stability, resistance to fibrillation	Surface coating, alteration of colloidal stability
Amino Acid-based ILs	Various Recombinant Proteins	Improved refolding yield, reduced inclusion body formation	Acting as chemical chaperones

The following diagram illustrates the multi-faceted mechanisms by which ILs protect proteins from physical and chemical degradation pathways.

Diagram 1: Mechanisms of protein stabilization by ionic liquids. ILs employ multiple simultaneous mechanisms to counteract specific degradation pathways.

Stabilization of Nucleic Acids

Nucleic acid-based therapeutics, including DNA and RNA, face significant delivery challenges due to their large molecular size, negative charge, and susceptibility to degradation by nucleases in biological environments [24]. Ionic liquids have shown remarkable potential in forming stable complexes with nucleic acids to overcome these barriers.

A key application is the use of ILs in non-viral gene delivery systems. Cationic ILs, particularly those with imidazolium or choline-based cations, can electrostatically condense nucleic acids into stable nanoparticles or polyplexes [15] [24]. This complexation offers a dual benefit: it compacts the nucleic acid into a more deliverable size and, crucially, protects it from nuclease degradation by creating a physical barrier that enzymes cannot easily penetrate. For example, studies have demonstrated that IL-coated lipid nanoparticles significantly increase the uptake of small interfering RNA (siRNA) into central nervous system targets, highlighting their potential for treating neurological disorders [24].

Furthermore, specific choline-based ILs have been engineered to function as multi-functional systems that not only protect nucleic acids from degradation but also enhance their cellular penetration. These ILs act as effective solubilizing agents for hydrophobic drugs while simultaneously stabilizing the nucleic acid structure, making them ideal candidates for combination therapies [24]. The table below summarizes key findings and applications.

Table 2: Efficacy of Ionic Liquids in Nucleic Acid Stabilization and Delivery

Ionic Liquid System	Nucleic Acid Type	Experimental Outcome / Protective Efficacy	Key Application
Imidazolium-based ILs	Plasmid DNA, siRNA	Formation of stable polyplexes; Significant protection from nuclease degradation	Non-viral gene delivery
Choline-based ILs	siRNA, mRNA	Enhanced nucleic acid stability; Improved cellular uptake and penetration	CNS delivery, vaccine technology
IL-coated Lipid Nanoparticles	siRNA	Increased siRNA uptake into CNS targets	Treatment of neurological diseases
Oil-in-IL Nanoemulsion	mRNA (Vaccine)	Improved stability of encapsulated mRNA; Enhanced immune response	Vaccine delivery platform

Experimental Protocols for Assessing Stability

To evaluate the protective efficacy of ILs on biopharmaceuticals, robust and quantitative experimental protocols are essential. Below are detailed methodologies for key assays cited in the literature.

Protocol: Quantifying Protein Degradation Using Modified SDS-PAGE

This protocol, adapted from a study on cleaning validation, provides a reliable method for quantifying the degree of protein degradation, which can be extended to assess the protective effect of ILs [43].

Objective: To quantify the extent of protein degradation (e.g., fragmentation or aggregation) in the presence and absence of ILs under stress conditions.

Materials and Reagents:

• SDS-PAGE System: BioRad Mini-PROTEAN Tetra System or equivalent.
• Gel Preparation Kit: Sigma-Aldrich kit for constructing 7.5% polyacrylamide (PAA) gels.
• Protein Sample: The biopharmaceutical protein of interest (e.g., monoclonal antibody).
• Stressing Agent: A chemical denaturant (e.g., 0.1M NaOH) or a source of oxidative stress.
• Ionic Liquid: The IL formulation being tested for its stabilizing property.
• Analysis Software: GelAnalyzer 2010 or equivalent gel image analysis software.

Methodology:

• Sample Preparation:
  • Prepare two sets of protein samples: one dissolved in a standard buffer and another in the same buffer containing the protective IL.
  • Subject both sets to identical stress conditions (e.g., incubation with 0.1M NaOH at room temperature for 10 minutes).
  • Crucially, omit the standard 90-95°C heating step in the SDS-PAGE sample preparation to avoid confounding the degradation caused by the stressor with that caused by sample heating [43].

• Gel Electrophoresis:

  • Construct 7.5% PAA gels. This concentration is optimal for high molecular weight proteins like antibodies [43].
  • Load the stressed and control samples onto the gel, including a molecular weight marker.
  • Run the electrophoresis according to standard protocols.

• Quantification and Analysis:

  • Use GelAnalyzer software to analyze the resulting gel.
  • Manually select gel lanes and bands to ensure accurate recognition of all protein species, including fragments and aggregates.
  • The software calculates the volume and molecular weight of protein bands based on the standards.
  • The degree of degradation is quantified by comparing the volume of the intact protein band in the stressed sample (with and without IL) to the unstressed control. A higher intensity of the intact band in the IL-protected sample indicates stabilization [43].

Protocol: Assessing Thermal Stability by Spectroscopic Methods

Objective: To determine the effect of ILs on the thermal stability of a protein, typically by measuring its melting temperature (Tm).

Materials and Reagents:

• Spectrofluorometer: Equipment capable of performing a thermal denaturation assay with a temperature-controlled cuvette holder.
• Fluorescent Dye: SYPRO Orange or equivalent environmentally sensitive dye.
• Protein Sample: Purified protein of interest.
• Ionic Liquid: The IL to be tested.

Methodology:

• Sample Preparation:
  • Prepare a solution of the protein in a suitable buffer.
  • Add the fluorescent dye, which fluoresces strongly upon binding to hydrophobic regions of the protein that become exposed during unfolding.
  • Prepare identical samples containing varying concentrations of the IL.

• Thermal Denaturation Assay:

  • Place the samples in the spectrofluorometer and set a temperature gradient (e.g., from 25°C to 95°C with a gradual increase of 1°C per minute).
  • Monitor the fluorescence intensity as a function of temperature.

• Data Analysis:

  • Plot the fluorescence intensity versus temperature to generate a denaturation curve.
  • The inflection point of this curve corresponds to the protein's melting temperature (Tm).
  • A higher Tm in the presence of the IL indicates a stabilizing effect, as more thermal energy is required to unfold the protein.

The following workflow visualizes the key steps in a stability assessment study, from formulation to data analysis.

Diagram 2: Experimental workflow for assessing biomolecule stability. This general workflow compares IL-protected samples against controls under stress conditions using various analytical methods.

The Scientist's Toolkit: Essential Research Reagents and Materials

Successful investigation into the stabilization of biopharmaceuticals using ILs requires a set of key reagents and analytical tools. The following table details essential components of the research toolkit.

Table 3: Key Research Reagent Solutions for IL-Biomolecule Stability Studies

Reagent / Material	Function and Role in Research	Example from Literature
Choline-Based ILs (e.g., Choline Geranate - CAGE)	Serves as a biocompatible IL platform for stabilizing proteins and enhancing penetration across biological barriers. Widely used in topical and transdermal delivery studies.	Clinical trials for rosacea and atopic dermatitis (NCT04886739, NCT05487963) [24].
Imidazolium-Based ILs (e.g., [C₄mim][BF₄])	Acts as a solvent or co-solvent to improve solubility of hydrophobic drugs and can stabilize protein structure against aggregation. Used in fundamental biophysical studies.	Studied for its effect on superfolder GFP aggregation and structure [15].
Amino Acid-Based ILs	Provides a low-toxicity, biodegradable IL option derived from natural sources. Used as a "chemical chaperone" to aid in protein refolding and prevent aggregation.	Employed to reduce the formation of inclusion bodies during recombinant protein production [15].
Modified SDS-PAGE System (e.g., BioRad Mini-PROTEAN)	A key analytical tool for separating and quantifying protein degradation products (fragments, aggregates) under non-heating conditions to accurately assess stability.	Used to quantify mAb degradation after caustic treatment [43].
Spectroscopic Assay Kits (e.g., SYPRO Orange)	Enable high-throughput measurement of protein thermal stability (Tm) by monitoring unfolding in real-time via fluorescence.	Standard method for determining the melting temperature of proteins in formulation screens.
Mass Spectrometry-Based Proteomics	Allows for deep, reproducible profiling of the proteome to identify off-target effects or changes in post-translational modifications caused by ILs.	Used in TPD research to measure degradation efficiency and kinetics [44].

Ionic liquids represent a paradigm shift in how we approach the stabilization of biopharmaceuticals. Their unmatched tunability allows them to be precisely engineered to counteract the specific physical and chemical degradation pathways of proteins, peptides, and nucleic acids. As research progresses, the future of ILs in biopharmaceuticals lies in the development of even smarter, biodegradable, and multifunctional materials [5]. Innovations such as stimuli-responsive ILs that release their payload in response to specific disease markers, and the integration of AI-driven design to rapidly screen optimal ion combinations for a given therapeutic, will further expand their potential [24]. While challenges in industrial scaling, long-term toxicity profiling, and regulatory approval remain, the continued translation of IL-based formulations into clinical trials signals a promising trajectory. ILs are poised to become key enablers of a new generation of stable, effective, and precisely delivered biologic medicines [5] [24].

Ionic liquids (ILs), a class of materials composed entirely of ions and typically liquid below 100 °C, have garnered significant interest in scientific and industrial fields due to their unique properties, including low volatility, excellent thermal stability, and strong solubilization power. [45] A key characteristic of ILs is their "designer solvent" nature, whereby their physicochemical properties can be finely tuned through structural design of the constituent cations and anions to fit specific application requirements. [45] This tunability provides an ideal platform for advanced applications in biomedicine, particularly in drug delivery systems where conventional solvents often face limitations.

The ionic nature of ILs creates unique solvation environments compared to conventional molecular solvents, facilitated by Coulombic forces combined with other weak intermolecular interactions such as hydrogen bonds, van der Waals forces, and π-π stacking. [45] These interactions enable microstructural diversification, including nano-segregated domains, making ILs particularly valuable for creating advanced functional materials. In the context of transdermal drug delivery, where the stratum corneum presents a formidable barrier to drug permeation, ILs offer promising solutions through their ability to enhance skin permeability and improve drug stability and bioavailability. [46] [47]

Among the various ILs investigated for biomedical applications, choline geranate (CAGE) has emerged as a leading candidate due to its exceptional biocompatibility and multifunctional capabilities. [47] As a deep eutectic solvent (DES) and/or ionic liquid formed at room temperature (RTIL), CAGE represents a promising material for active pharmaceutical ingredient (API) formulations, combining the favorable safety profiles of its components with enhanced drug delivery performance. [47]

CAGE: Synthesis, Properties, and Bioactive Potential

Synthesis and Physicochemical Properties

CAGE is synthesized using Generally Recognized as Safe (GRAS) reagents present in the FDA list: choline bicarbonate and geranic acid. [47] Choline is a water-soluble crucial nutrient present in the liver and in phospholipids redundant in cell membranes, while geranic acid occurs in lemon grass and is used as a flavoring agent. [47] The synthesis involves a salt metathesis reaction, with the specific ratio of choline to geranic acid determining whether the product is classified as a room temperature ionic liquid (RTIL) or a deep eutectic solvent (DES).

Table 1: Synthesis Variations and Classification of CAGE

Choline:Geranic Acid Ratio	Classification	Key Characteristics
1:1	Room Temperature Ionic Liquid (RTIL)	Organic salts with 1:1 component ratio of cations and anions
1:2	Deep Eutectic Solvent (DES)	Prepared by varying ratios of cation of charged or neutral species; most effective for transdermal delivery

The synthesis protocol involves mixing choline bicarbonate and geranic acid with methanol until CO₂ production ceases, followed by solvent removal using a rotary evaporator at 60°C. [48] The prepared CAGE is then transferred to a sealed container with nitrogen bubbling to prevent oxidation. [48] Research has demonstrated that a molar ratio of 1:2 of choline to geranic acid yields the highest transdermal delivery enhancement, establishing this formulation as optimal for drug delivery applications. [49]

The physicochemical properties of CAGE vary with its composition. The most effective 1:2 ratio CAGE has a viscosity of 569 ± 19 mPa·s, conductivity of 13.79 ± 0.28 mS·m⁻¹, with a diffusion coefficient of 2.2 × 10⁻¹² m²·s⁻¹. [47] Additionally, CAGE has a molecular weight of 440.32 g·mL⁻¹ with a density of 0.989 ± 0.001 g·mL⁻¹ at 25°C. [47] These properties contribute to its exceptional performance in transdermal drug delivery systems.

Bioactive Potential and Antimicrobial Mechanisms

CAGE exhibits significant broad-spectrum bioactivity owing to its biologically active precursors. It has demonstrated potent antimicrobial activities against various pathogens, including bacteria, fungi, and viruses. [47] Laboratory studies have confirmed its efficacy against clinically isolated strains of Candida albicans, Staphylococcus aureus, Mycobacterium tuberculosis, and laboratory strains of herpes simplex virus. [47]

The antimicrobial mechanism of CAGE involves disruption of microbial membrane integrity. Fluorescence flow cytometry studies have demonstrated that CAGE causes loss of cell membrane integrity through rupture, while FTIR spectroscopy has revealed alterations in membrane lipid profiles associated with antibacterial resistance. [47] Molecular modeling and simulation studies indicate that chionine efficiently screens the negative charge on bacterial membranes, facilitating the penetration of geranate or geranic acid into the membrane structure. [47] This dual mechanism explains CAGE's exceptional potency against various microorganisms.

CAGE has shown remarkable effectiveness against bacterial biofilms, achieving efficient elimination at low concentrations (3.65 mM) within just 2 hours. [47] When applied to one-day-old Staphylococcus aureus biofilms, a 0.1% CAGE solution reduced biofilm vulnerability by three orders of magnitude in merely 15 minutes while simultaneously rupturing the outer polymeric layer of the biofilms. [47] This potent antibiofilm activity, combined with its skin penetration capability, enables CAGE to treat deep skin infections such as those caused by Propionibacterium acnes. [47]

CAGE Antimicrobial Mechanism

CAGE as a Transdermal Permeation Enhancer: Mechanisms and Design Principles

Mechanism of Skin Barrier Modulation

The stratum corneum, the outermost layer of the epidermis, represents the primary barrier to transdermal drug delivery due to its structure of corneocytes embedded in a lipid matrix, which significantly restricts permeation of hydrophilic and high-molecular-weight molecules. [46] CAGE enhances transdermal delivery by causing transient alterations in the intercellular and intracellular lipids and protein organization of the stratum corneum. [48] This disruption allows enhanced penetration of bioactive molecules through the skin layers.

Research has revealed that the potency of ILs in enhancing transdermal drug delivery correlates inversely with the inter-ionic interactions, as determined by 2D NMR spectroscopy. [49] This understanding provides a fundamental design principle for optimizing ILs for skin permeability enhancement. Compared to traditional organic solvents like ethanol, CAGE is less toxic to cells, minimizing problems associated with solvent-induced skin irritation while providing superior permeation enhancement. [48]

Design Principles for Optimal Transdermal Enhancement

Systematic studies examining the relationship between IL structure and skin penetration enhancement have identified critical factors influencing efficacy. Assessment of ion stoichiometry using CAGE demonstrated that a molar ratio of 1:2 of choline to geranic acid yields the highest drug delivery. [49] Subsequent preparation of CAGE variants using anions with structural similarity to geranic acid and cations with structural similarity to choline at this optimal ratio enabled researchers to establish clear structure-activity relationships.

The fundamental insight from these investigations revealed that weaker inter-ion interactions within the ionic liquid structure correspond to greater potency in enhancing transdermal drug delivery. [49] This understanding enabled the rational design of new ILs with maximized transdermal enhancement capability, with one newly designed IL providing the highest delivery of ruxolitinib of all ILs tested in comprehensive studies. [49] This systematic approach provides a generalized framework for optimizing ILs for enhancing skin permeability.

Table 2: Key Design Principles for Transdermal Ionic Liquids

Design Parameter	Optimal Characteristic	Impact on Transdermal Delivery
Ion Stoichiometry	1:2 choline:geranic acid	Highest skin penetration enhancement
Inter-ionic Interactions	Weak Coulombic forces	Enhanced skin permeability
Cation Structure	Similar to choline	Biocompatibility and efficacy
Anion Structure	Similar to geranic acid	Membrane disruption capability
Physical State	Liquid at room temperature	Practical application

Experimental Protocols and Research Methodologies

CAGE Synthesis Protocol

Materials:

• Geranic acid (85% stabilized; CAS No. 459-80-3)
• Choline bicarbonate (CAS No. 62-49-7)
• Methanol (HPLC-grade, CAS No: 67-56-1)
• Round-bottom flask (1000 mL)
• Rotary evaporator
• Nitrogen gas source

Procedure:

• Add 48 mL of geranic acid to a 1000 mL round-bottom flask.
• Add 20 mL of choline bicarbonate at 80% (w/v).
• Add 20 mL of methanol to the mixture.
• Mix continuously until CO₂ production ceases completely.
• Transfer the mixture to a rotary evaporator and remove solvent at 60°C for approximately 20 minutes.
• Transfer the prepared CAGE to a 50 mL Falcon tube.
• Bubble nitrogen through the CAGE to scavenge dissolved oxygen and prevent oxidation.
• Cap the tube and seal with Parafilm for storage. [48]

Formulation of Biopolysaccharide Gels with CAGE for Transdermal Delivery

Materials:

• Locust bean gum (LBG)
• Curcumin (95% purity)
• Methylparaben
• Ultrapure water
• CAGE (1:2 ratio)
• Magnetic stirrer

Procedure for Gel with Curcumin and CAGE:

• Disperse precise mass concentrations of curcumin (2.087%, w/w), methylparaben (0.1%, w/w), and locust bean gum (2%, w/w) in ultrapure water under continuous magnetic stirring.
• Add synthesized CAGE at the desired concentration (typically 1-5% v/v) to the mixture.
• Continue stirring until a homogeneous gel forms.
• Transfer to appropriate containers for storage and application. [48]

In Vivo Permeation and Efficacy Assessment

Animal Model:

• Mice of BALB/c strain (7-9 weeks old, average weight 25 g)
• Psoriasis induction using imiquimod (IMQ) cream

Experimental Design:

• Induce psoriasis-like lesions by applying imiquimod cream to mouse skin.
• Apply developed gel formulations containing curcumin and CAGE to affected areas.
• Monitor histological changes through skin biopsies.
• Evaluate reversal of psoriasis manifestations compared to control treatments. [48]

Assessment Parameters:

• Erythema (redness) reduction
• Skin thickening measurement
• Epidermal changes (acanthosis, parakeratosis)
• Inflammatory infiltrate composition
• Comparison to normal skin histology [48]

CAGE Experimental Workflow

Research Reagent Solutions and Essential Materials

Table 3: Essential Research Reagents for CAGE Studies

Reagent/Material	Function/Purpose	Application Context
Choline bicarbonate	Cation source for CAGE synthesis	IL synthesis
Geranic acid (85% stabilized)	Anion source for CAGE synthesis	IL synthesis
Locust bean gum (LBG)	Gelling agent for formulations	Biopolysaccharide gel preparation
Curcumin (95% purity)	Model hydrophobic drug compound	Transdermal delivery studies
Methylparaben	Preservative in gel formulations	Prevention of microbial growth
Imiquimod cream	Psoriasis induction in mouse model	In vivo disease model creation
Methanol (HPLC-grade)	Solvent for synthesis	CAGE preparation
Franz diffusion cells	Assessment of drug permeation	In vitro transdermal studies

Applications and Efficacy Data for Diverse APIs

Small Molecule Delivery: Curcumin for Psoriasis Treatment

Research has demonstrated CAGE's remarkable efficacy in enhancing the transdermal delivery of curcumin for psoriasis treatment. A study developing a biopolysaccharide hydrogel formulation integrating curcumin with CAGE as a permeation facilitator showed high potential for psoriasis applications, reversing histological manifestations to a state very close to normal skin. [48] The prepared gel containing curcumin and CAGE-IL demonstrated significant therapeutic effects in imiquimod-induced psoriasis in mice, highlighting CAGE's ability to enhance permeation of relatively hydrophobic molecules like curcumin that normally face challenges crossing the stratum corneum. [48]

Broad-Spectrum API Delivery Enhancement

CAGE has demonstrated versatility in enhancing skin permeation across diverse API types with differing hydrophilicities. Studies investigating the transdermal delivery of model drugs with varying properties, including acarbose and ruxolitinib, confirmed CAGE's effectiveness for both hydrophilic and hydrophobic compounds. [49] This broad-spectrum enhancement capability positions CAGE as a versatile platform technology for transdermal drug delivery beyond specific molecular classes.

Additional research has documented CAGE's efficacy in facilitating the skin permeation of various challenging substances, including insulin and bacteriophage particles, further attesting to its capability with macromolecular and complex biological therapeutics. [48] The demonstrated ability to enhance permeation of molecules ranging from small hydrophobic drugs to large proteins underscores CAGE's potential to expand the scope of transdermally deliverable therapeutics.

Table 4: Efficacy of CAGE in Enhancing Transdermal Delivery of Various APIs

API/Drug	Molecular Characteristics	Delivery Enhancement	Therapeutic Application
Curcumin	Hydrophobic, low solubility	Reversed psoriasis manifestations	Psoriasis treatment
Ruxolitinib	Varies with formulation	Highest delivery with optimized IL	Under investigation
Insulin	Macromolecular protein	Facilitated skin permeation	Diabetes management
Bacteriophage particles	Complex biological structures	Enhanced transdermal delivery	Antibacterial therapy
Acarbose	Hydrophilic character	Significant permeation enhancement	Diabetes treatment

CAGE represents a paradigm shift in transdermal drug delivery, offering a biocompatible, multifunctional platform that addresses fundamental challenges in percutaneous absorption. Its unique combination of properties—including tunable physicochemical characteristics, broad-spectrum antimicrobial activity, and potent permeation enhancement—positions it as a versatile tool for advancing transdermal therapeutics. The established design principles for optimizing ILs for skin permeability provide a systematic framework for further development of next-generation transdermal delivery systems.

Future research directions should focus on expanding the application of CAGE to additional therapeutic classes, particularly biologics and macromolecules that currently face significant delivery challenges. Additionally, continued investigation into the long-term safety profile and potential for commercial scalability will be essential for clinical translation. As the field of ionic liquid research advances, complemented by emerging technologies like machine learning for property prediction, [50] the rational design of IL-based transdermal systems promises to unlock new possibilities in non-invasive drug delivery, ultimately improving patient compliance and therapeutic outcomes across a broad spectrum of medical conditions.

Evidence and Advantage: Validating Efficacy and Comparing ILs to Conventional Systems

Ionic liquids (ILs), a class of salts with melting points below 100°C, are emerging as powerful and versatile tools in pharmaceutical research and development. Their appeal lies in their status as "designer solvents," meaning their physicochemical properties—including solubility, polarity, viscosity, and thermal stability—can be precisely tailored by selecting and modifying their cationic and anionic components [51] [52]. This tunability allows researchers to engineer ILs for specific biological applications, from enhancing drug permeability across biological barriers to improving the stability of therapeutic compounds. Within the context of preclinical drug development, ILs are being innovatively applied to overcome significant challenges in the treatment of major diseases. This whitepaper details three specific preclinical success stories where IL-based strategies have demonstrated remarkable efficacy in disease models for diabetes, skin cancer, and fungal infections, showcasing their potential to advance novel therapeutic paradigms.

Ionic Liquids in Diabetes Research: Targeting Aldose Reductase

Diabetic complications, such as neuropathy and cataracts, are driven by the activation of the polyol pathway under hyperglycemic conditions. Aldose reductase (ALR2), a key enzyme in this pathway, converts glucose to sorbitol, leading to osmotic stress and oxidative damage. While ALR2 is a validated drug target, many existing inhibitors have been limited by adverse side effects, creating a need for new therapeutic leads [53].

Preclinical Success with Coumarin-Linked Schiff Bases

A 2025 study demonstrated a sustainable and effective approach for synthesizing new coumarin-linked Schiff bases using a DABCO-based ionic liquid (DABCO-C7-F) as a reaction medium [53]. This method was not only cost- and time-effective but also successfully generated a library of novel compounds with anti-diabetic potential.

The synthesized analogues were evaluated for their ability to inhibit ALR2. The results were compelling, with compounds exhibiting IC50 values ranging from 1.61 to 11.20 µM, indicating potent inhibition. A critical aspect of this success was the high selectivity these compounds showed for ALR2 over the related ALR1 enzyme, which is desirable for minimizing off-target effects [53]. Molecular docking studies elucidated that the active compounds fit snugly into the enzyme's active site, forming stable interactions that explain the observed inhibitory activity and selectivity.

Table 1: Efficacy of Select Coumarin-Linked Schiff Bases as Aldose Reductase Inhibitors

Compound Analogue	IC50 against ALR2 (µM)	Selectivity (ALR2 vs. ALR1)	Key Finding
Lead Compound	1.61 µM	High	Potent and selective ALR2 inhibition [53]
Other Active Analogues	≤ 11.20 µM	High	Library demonstrated consistent activity [53]

Detailed Experimental Protocol

• Synthesis in Ionic Liquid: The coumarin-linked Schiff bases were synthesized by condensing coumarin aldehydes with appropriate amines directly in the DABCO-C7-F ionic liquid. The IL acted as a green and efficient catalyst and solvent [53].
• In Vitro Enzyme Inhibition Assay: The inhibitory activity against purified human ALR2 and ALR1 enzymes was measured. The assay mixture typically contained the enzyme, NADPH as a cofactor, the substrate (e.g., glyceraldehyde), and the test compound. The rate of NADPH consumption, monitored spectrophotometrically at 340 nm, indicates enzyme activity. IC50 values are determined by measuring this rate in the presence of varying concentrations of the inhibitor [53].
• Molecular Docking: To understand the binding mode, the most active compounds were computationally docked into the crystal structure of ALR2 using software like AutoDock Vina. The docking poses were analyzed for hydrogen bonding, hydrophobic interactions, and other stabilizing forces within the active site [53].

Diagram 1: IL compound inhibits ALR2 in the polyol pathway.

Ionic Liquids in Skin Cancer Therapy: Targeted Transdermal Delivery

Melanoma is the most lethal form of skin cancer, accounting for a disproportionate number of skin cancer deaths. Dacarbazine (DTIC) is a standard chemotherapy drug for advanced melanoma, but its efficacy is limited, and it causes systemic side effects like bone marrow suppression and gastrointestinal reactions. Transdermal delivery offers a way to bypass these issues, but the skin's stratum corneum presents a formidable barrier, and water-soluble drugs like DTIC penetrate it poorly [54] [55].

Preclinical Success with an Ionic Liquid Nanoemulsion

A groundbreaking 2025 study addressed these challenges by developing a novel ionic liquid nanoemulsion transdermal delivery system (HIT-1/PM-MEs) for targeted melanoma therapy [54] [55]. The strategy was twofold: first, a new drug candidate (HIT-1) was synthesized by conjugating DTIC with a benzothiocycloheptane derivative; second, this conjugate was formulated into an oil-in-oil ionic liquid nanoemulsion.

The results were striking. The lead compound HIT-1 itself showed 10.3-fold stronger cytotoxicity against B16-F10 melanoma cells (IC50 = 7.86 µM) than DTIC alone. When delivered via the optimized ionic liquid nanoemulsion (HIT-1/PM-ME-1-1), the system was 11.8-fold more potent than DTIC [54] [55]. In vivo studies in melanoma models confirmed that this transdermal system achieved optimal antitumor effects, induced apoptosis, and stimulated an antitumor immune response, providing a comprehensive and effective therapeutic strategy.

Table 2: Preclinical Efficacy of Ionic Liquid Nanoemulsion for Melanoma

Parameter	Dacarbazine (DTIC)	HIT-1 Compound	HIT-1/PM-ME-1-1 Formulation
Cytotoxicity (IC50) on B16-F10 cells	Baseline	10.3-fold stronger than DTIC [55]	11.8-fold stronger than DTIC [54]
Cellular Mechanism	-	Induces apoptosis & G0/G1 phase arrest [55]	Regulates p53, Bcl-2, Cleaved-caspase 3 [55]
In Vivo Effect	-	-	Optimal antitumor effect & immune stimulation [54]

Detailed Experimental Protocol

• Synthesis of HIT-1 Conjugate: The benzothiocycloheptane skeleton was structurally modified and coupled with DTIC using a multi-step synthetic route, culminating in the final product HIT-1 [54].
• Preparation of Ionic Liquid Nanoemulsion (HIT-1/PM-MEs): The formulation was a self-assembled oil-in-oil nanoemulsion. It utilized a biocompatible ionic liquid (L-pyrrolidone carboxylic acid-matrine, P-M IL) as a carrier, combined with emulsifiers Tween-80 and SPAN-80. E-TPGS micelles were incorporated to confer pH-sensitive tumor-targeting properties [54].
• In Vitro Cytotoxicity and Transdermal Assay: Cytotoxicity was evaluated via MTT assay on B16-F10 melanoma cells. Transdermal permeation was studied using Franz diffusion cells with excised skin, demonstrating the nanoemulsion's superior ability to deliver the drug through the stratum corneum [54].
• In Vivo Efficacy Study: A murine melanoma model (e.g., B16-F10 tumor-bearing mice) was used. The HIT-1/PM-ME-1-1 formulation was applied topically, and tumor volume, growth inhibition, and biomarkers of immune response were monitored over time [54].

Diagram 2: IL nanoemulsion mechanism for transdermal therapy.

Ionic Liquids and Antifungal Research: Addressing a Growing Threat

Fungal infections represent a significant and growing global health burden, causing over 3.8 million deaths annually. The situation is exacerbated by the emergence of resistant strains, the limited arsenal of antifungal drug classes, and the fact that fungal cells are eukaryotic, making it difficult to find selective drug targets that do not also harm human cells [56]. While research into direct ionic liquid antifungals is still emerging, IL-based strategies are being explored to improve drug formulations and delivery.

Preclinical Investigations and Alternative IL-Inspired Approaches

Although the search results did not provide a specific preclinical story of an IL-based small molecule antifungal, they highlight the serious challenges and innovative approaches in the field. The major classes of antifungals—polyenes, azoles, echinocandins, pyrimidine analogs, and allylamines—are all constrained by issues of resistance, toxicity, and sometimes poor bioavailability [56]. This underscores the critical need for new delivery systems that can enhance the efficacy and safety of existing drugs, an area where ionic liquids' tunable properties could be highly advantageous.

Concurrently, research into other non-conventional liquid-based therapies is advancing. A 2025 study on Plasma-Activated Liquids (PALs) demonstrated their efficacy against Candida albicans in both planktonic and biofilm forms. Argon-activated saline and distilled water achieved approximately a 50% reduction in fungal viability and retained significant antibiofilm activity, all while being non-toxic to mammalian cells [57]. This illustrates the broader trend of exploring activated liquid systems to combat resistant fungal infections.

Detailed Experimental Protocol for Antifungal Testing

The following protocol is representative of standard preclinical antifungal evaluation, which would be applicable for testing future IL-based antifungal formulations.

• Broth Microdilution for Minimum Inhibitory Concentration (MIC): This CLSI-standard method involves preparing serial dilutions of the test compound in a broth medium in a 96-well plate. A standardized inoculum of the fungal strain (e.g., C. albicans, C. auris) is added to each well. The plate is incubated, and the MIC is identified as the lowest concentration that visually inhibits fungal growth [56].
• Biofilm Susceptibility Assay: Fungal biofilms are grown on a surface (e.g., polystyrene) for 24-48 hours. The mature biofilms are then treated with the test compound. Metabolic activity of the remaining viable cells in the biofilm is quantified using assays like XTT or resazurin, which measure cellular reduction potential [57].
• Cytotoxicity Assay: To determine selectivity, the cytotoxic effect of the compound is evaluated on mammalian cell lines (e.g., Vero cells). An MTT or similar assay is performed, and the concentration that reduces cell viability by 50% (CC50) is calculated. A high CC50/MIC ratio indicates a good safety window [57].

Table 3: Major Antifungal Classes and Their Challenges

Antifungal Class	Target / Mechanism	Key Challenges & Resistance
Azoles	Inhibit ergosterol synthesis [56]	Widespread resistance; drug-drug interactions [56]
Echinocandins	Inhibit cell wall β-glucan synthesis [56]	Intravenous administration only [56]
Polyenes	Bind to ergosterol, disrupting membrane [56]	Significant toxicity (e.g., nephrotoxicity) [56]
Allylamines	Inhibit ergosterol synthesis [56]	Emerging resistance [56]
Pyrimidine Analogs	Interfere with nucleic acid synthesis [56]	Limited spectrum; growing resistance [56]

The Scientist's Toolkit: Key Research Reagents and Materials

The following table compiles essential materials and reagents used in the featured preclinical studies, providing a resource for researchers aiming to work in this domain.

Table 4: Research Reagent Solutions for Ionic Liquid-Based Preclinical Studies

Reagent / Material	Function / Application	Example from Research
DABCO-based IL (e.g., DABCO-C7-F)	Green solvent & catalyst for organic synthesis	Synthesis of coumarin-linked Schiff bases for diabetes [53]
L-pyrrolidone carboxylic acid-matrine IL	Biocompatible carrier for transdermal delivery	Core component of the HIT-1 nanoemulsion for melanoma [54]
Tween-80 & SPAN-80	Non-ionic surfactants for emulsion stabilization	Emulsifiers in the ionic liquid nanoemulsion system [54]
D-α-tocopheryl polyethylene glycol succinate (TPGS)	Emulsifier & permeability enhancer for targeted delivery	Imparted pH-sensitivity for tumor targeting in nanoemulsion [54]
Choline-based ILs (e.g., Choline Geranate)	Permeation enhancer for transdermal delivery	Promotes skin delivery of various drugs (e.g., curcumin) [52]

The preclinical success stories highlighted in this whitepaper underscore the transformative potential of ionic liquids in modern drug development. By leveraging the tunable properties of ILs, researchers have made significant strides in overcoming persistent challenges in diabetes, oncology, and infectious disease. From enabling the sustainable synthesis of potent new enzyme inhibitors to engineering sophisticated transdermal delivery systems that target aggressive skin cancers, ILs have proven to be more than just inert solvents; they are active and integral components of therapeutic strategies. As research continues to expand, particularly into complex areas like antifungal therapy, the ability to design task-specific ionic liquids promises to unlock further innovative treatments, ultimately paving the way for more effective, targeted, and patient-friendly medicines.

Ionic Liquids (ILs) are a class of salts characterized by their low melting points (often below 100 °C), composed of large, asymmetric organic cations and organic or inorganic anions [2]. Their most defining feature in pharmaceutical and materials science is their highly tunable nature; by selecting different combinations of cations and anions, researchers can precisely engineer IL properties such as polarity, hydrophilicity/hydrophobicity, viscosity, and solvent miscibility for specific applications [58] [2]. This tunability positions ILs as "designer solvents" capable of addressing persistent challenges in drug delivery, particularly the poor solubility and low permeation of many active pharmaceutical ingredients (APIs) [24].

The evolution of ILs is categorized into generations. First-generation ILs focused on unique physical properties but often exhibited toxicity and poor biodegradability. Second-generation ILs offered enhanced stability and tunable chemical properties. The most significant for biomedical applications are third-generation ILs, which incorporate biologically active or benign ions, such as choline and amino acids, resulting in low toxicity, good biodegradability, and tailored biocompatibility [7] [19] [5]. This progression has unlocked the potential for ILs to serve as superior alternatives to conventional organic solvents like acetone, ethanol, and dimethyl sulfoxide (DMSO), which are plagued by issues of volatility, toxicity, and limited ability to enhance drug delivery [19] [24].

Superior Solubilization Capacity of Ionic Liquids

Mechanisms of Solubility Enhancement

The exceptional ability of ILs to dissolve poorly soluble compounds stems from their unique mechanism of action, which goes beyond the capabilities of traditional organic solvents. The primary mechanisms include:

• Disruption of Molecular Crystals: ILs can effectively break the strong crystal lattice of APIs through powerful ion-dipole interactions and hydrogen bonding. The ions within the IL interact with the functional groups of the drug molecule, disrupting the cohesive energy of the crystal and facilitating its dissolution [24].
• Hydrogen Bonding Network: Unlike conventional solvents, ILs function as three-dimensional hydrogen-bonded networks. Both the cation and anion can participate in extensive hydrogen bonding with solute molecules, providing a multifaceted solvation environment that can be tailored to a specific API [59] [2].
• Formation of API-Ionic Liquids (API-ILs): A paradigm-shifting strategy involves converting an acidic or basic drug molecule itself into an ionic liquid by pairing it with a benign counterion. This approach can transform a solid, crystalline drug into a liquid salt at room temperature, fundamentally eliminating solubility barriers associated with the crystal lattice and potentially increasing solubility by several orders of magnitude [7] [24].

Quantitative Comparison of Solubilization Efficacy

The following table summarizes experimental data demonstrating the enhanced solubility of various drugs in IL-based systems compared to conventional solvents and water.

Table 1: Solubility Enhancement of Drugs using Ionic Liquids

Drug Compound	IL Formulation	Conventional Solvent/Water Solubility	IL-Based Solubility	Enhancement Factor	Reference
Ketoconazole	IL-based formulation	Limited solubility in water	Significant improvement for treating T. interdigitale infection	Not Specified	[24]
Ibuprofen	[Proline ethylester][Ibuprofen] (API-IL)	Poor aqueous solubility	Exists primarily in neutral form, enhancing permeation	Not Specified	[58]
Navitoclax (NAVI)	[Choline][Octanoate] (COA)	Low aqueous solubility	Improved skin penetration and retention in deeper skin layers	Not Specified	[58]
Tretinoin	[Choline][Tretinoin] (API-IL)	Limited solubility	High skin permeability due to tailored lipophilicity	Not Specified	[58]
Cellulose	1-Ethyl-3-methylimidazolium acetate ([EMIM][Ac])	Insoluble in water & most organic solvents	>20 wt% solubility	Dramatic (From 0 to soluble)	[60]
Curcumin	[C₄C₁im][N(Tf)₂]	Low solubility in water	Efficient solvent for esterification reaction (98% yield)	Not Specified	[2]

Enhanced Permeation and Drug Delivery Capabilities

Mechanisms of Permeation Enhancement

A principal challenge in drug delivery is overcoming biological barriers, such as the skin's stratum corneum (SC) or the intestinal epithelium. ILs enhance drug permeation through several distinct mechanisms, which are illustrated in the following workflow and described in detail below.

Diagram 1: IL Permeation Enhancement Mechanisms

• Lipid Disruption and Fluidization: ILs, particularly those with amphiphilic ions, can integrate into the lipid bilayers of biological membranes like the stratum corneum. This integration increases the fluidity of the lipid domains, reducing the barrier's structural integrity and facilitating the diffusion of drug molecules [58] [24].
• Lipid Extraction and Pore Formation: Certain ILs can extract lipids from the membrane, creating transient pores or pathways. For instance, choline-geranic acid IL (CAGE) has been shown to extract skin lipids, replacing them with IL and water, which enables faster diffusion of molecules, including large entities like dextran [58].
• Interaction with Keratin and Barrier Reduction: ILs can disrupt the dense network of corneocytes in the SC by interacting with and altering the conformation of keratin proteins. This action reduces the overall barrier resistance of the skin, further promoting drug permeation [58].

Quantitative Analysis of Permeation Enhancement

The efficacy of ILs in improving drug permeation is quantitatively demonstrated in numerous studies, as summarized in the table below.

Table 2: Permeation Enhancement of Drugs using Ionic Liquids

Drug / Active Compound	Biological Barrier	IL Formulation	Key Permeation Finding	Reference
Dextran	Skin (Stratum corneum)	[Choline][Geranic Acid] (CAGE)	Induces lipid extraction, facilitating faster diffusion	[58]
NSAIDs (e.g., Ibuprofen)	Skin (Stratum corneum)	Ethylamine-based API-ILs	Causes conformational disturbances and phase changes in the lipid bilayer	[58]
Peptides	Skin (Stratum corneum)	[Choline][Fatty Acid]	Permeation occurs through the intracellular lipids of the SC	[58]
Insulin	Skin	Choline-geranic acid IL (CAGE)	Enables transdermal delivery of macromolecules	[58] [24]
siRNA	Skin	Composite IL	Effective for treatment of psoriasis-like lesions	[24]
Vancomycin HCl	Skin	Choline-based ionogel	Promising transdermal delivery achieved	[52]
Fn14 siRNA	Skin	Novel composite IL	Successful topical delivery for psoriasis treatment	[58]

Experimental Protocols for Key Applications

Protocol 1: Formulating an API-Ionic Liquid

This protocol outlines the synthesis of an API-IL through a simple metathesis or acid-base neutralization reaction [7] [19].

• Selection of Ion Pairs: Identify an ionizable API (e.g., a weak acid or base). Select a biocompatible counterion (e.g., choline for an acidic API, or geranic acid for a basic API).
• Reaction Setup: For an acidic API, dissolve a stoichiometric amount of choline bicarbonate in a minimal volume of methanol. Slowly add the acidic API to the solution with stirring.
• Ion Exchange and Synthesis: Allow the acid-base reaction to proceed for 12-24 hours at room temperature or with mild heating (e.g., 40°C) to facilitate ion exchange and formation of the API-IL.
• Purification: Remove the solvent (methanol) and any volatile by-products (e.g., CO₂) under high vacuum. The resulting liquid or low-melting-point solid is the purified API-IL.
• Characterization: Confirm the structure and purity using techniques such as ¹H-NMR and FT-IR. Analyze the thermal properties via Differential Scanning Calorimetry (DSC) to confirm the absence of a sharp melting point [19].

Protocol 2: Evaluating Transdermal Permeation Using Franz Diffusion Cell

This standard protocol is used to quantify the permeation enhancement of a drug formulated with an IL [58].

• Membrane Preparation: Use excised human or porcine skin, with the hypodermis carefully removed. The skin membrane is mounted between the donor and receptor compartments of the Franz diffusion cell.
• Formulation Application: Apply a precise dose of the drug-loaded IL formulation (e.g., IL solution, IL-based gel, or API-IL itself) to the donor side of the skin membrane.
• Sampling: The receptor compartment is filled with a suitable buffer (e.g., phosphate-buffered saline, PBS) maintained at 37°C with constant stirring. At predetermined time intervals, aliquot samples are withdrawn from the receptor compartment and replaced with fresh buffer.
• Analysis: The concentration of the drug in each sample is quantified using a validated analytical method, typically High-Performance Liquid Chromatography (HPLC).
• Data Processing: Cumulative drug permeation per unit area is plotted against time. Key parameters like the flux (Jss) and enhancement ratio (flux with IL / flux with control formulation) are calculated to objectively evaluate the IL's performance [58].

The Scientist's Toolkit: Essential Research Reagents and Materials

Table 3: Key Reagents and Materials for IL-Based Formulation Research

Reagent/Material	Function/Application	Example in Research
Choline Bicarbonate / Chloride	Biocompatible cation precursor for synthesizing third-generation ILs.	Used to create choline-geranate (CAGE) for transdermal delivery [19] [24].
Amino Acids (e.g., Glycine, Proline)	Serve as anions to form biocompatible, low-toxicity Bio-ILs.	Choline-glycine IL for drug solubilization and antimicrobial studies [19].
Geranic Acid	A natural, bioactive anion that contributes to permeation enhancement.	Key component of CAGE, a well-studied IL for transdermal delivery of small and large molecules [58] [24].
1-Butyl-3-methylimidazolium ([C₄mim]) salts	Versatile, widely studied cations for first/second-generation ILs; useful for fundamental solubility studies.	[C₄mim][PF₆] used in mixtures with organic solvents like benzonitrile for solubility and structural studies [61].
Franz Diffusion Cell System	Essential experimental apparatus for in vitro assessment of transdermal drug permeation kinetics.	Used to quantify the enhancement ratio of drug permeation facilitated by ILs [58].

The evidence overwhelmingly demonstrates that IL-based formulations fundamentally outperform conventional organic solvents in enhancing both the solubility and permeation of challenging drug candidates. The core of this superiority lies in the tunable properties of ILs, which allow researchers to design solvents that not only dissolve robust crystal lattices but also reversibly modulate the properties of biological barriers. The emergence of third-generation Bio-ILs and API-ILs effectively addresses earlier concerns regarding toxicity and biodegradability, paving the way for their clinical translation. As research progresses, the integration of computational design and sustainable chemistry principles with IL technology promises to unlock even more sophisticated and targeted drug delivery solutions, solidifying the role of ILs as indispensable tools in modern pharmaceuticals.

Ionic Liquids (ILs) are organic salts with melting points below 100°C, characterized by their unique and tunable physicochemical properties [62]. This tunability, achieved by combining different cations and anions, makes them powerful tools in pharmaceutical research. ILs can be engineered to enhance drug solubility, stability, and permeability, thereby addressing key challenges in drug development [62]. The evolution of ILs has progressed to a third generation, which emphasizes biocompatibility by using ions derived from natural, renewable sources, such as choline and amino acids, to reduce toxicity and improve safety profiles [62]. This review examines the progress of IL-based formulations along the clinical translation pathway, highlighting the milestones from preclinical discovery to active clinical trials.

Preclinical Evidence for IL Formulations

Key Advancements in Preclinical Formulations

Preclinical research has successfully demonstrated the potential of ILs to solve formulation challenges, particularly for drugs with poor bioavailability. The table below summarizes key preclinical studies.

Table 1: Summary of Key Preclinical IL Formulations

IL Formulation	API/Drug Model	Key Finding	Reference
Choline-Arginine (ChArg)	IgG (Monoclonal Antibody)	Enabled ultra-high concentration (>200 mg/mL) formulations with low viscosity (<20 cP) and enhanced subcutaneous bioavailability.	[63]
Lipophilic API-IL (with Docusate)	Metformin	Significantly enhanced gastrointestinal permeability (2.3 to 6.3-fold increase) in ex vivo rat intestinal models.	[64]
Acetylcholine-Hexenoate	Rituximab	Enhanced subcutaneous bioavailability by ~200% at a low mAb concentration (10 mg/mL).	[63]
Choline-based Bio-ILs	Various small/large molecules	Improved transdermal delivery and demonstrated high biodegradability and low toxicity.	[62]

Experimental Protocols in Preclinical IL Research

The following experimental designs are critical for generating robust preclinical data on IL formulations.

Table 2: Key Experimental Protocols for Preclinical IL Evaluation

Protocol Objective	Core Methodology	Key Measurements & Analyses
Formulation Stability & Viscosity Assessment	1. Synthesize ILs via salt metathesis or neutralization reactions.2. Dissolve the Active Pharmaceutical Ingredient (API) in the IL.3. Subject the formulation to accelerated stability conditions (e.g., 40°C, 75% relative humidity).	- Viscosity: Measured using a rheometer.- Aggregation: Analyzed via size-exclusion chromatography (SEC-HPLC).- Structural Integrity: Confirmed by circular dichroism (CD) spectroscopy.
In Vitro & Ex Vivo Permeability Studies	1. Formulate the drug into an IL (e.g., API-IL).2. For ex vivo studies, use tissue mucosae (e.g., rat intestine) mounted in Ussing chambers.3. Measure the transport of the API across the membrane over time.	- Apparent Permeability (Papp): Calculated from the cumulative amount of drug transported.- Enhancement Ratio: Fold-increase in Papp compared to a control formulation (e.g., API hydrochloride).
In Vivo Biodistribution & Efficacy	1. Administer the IL-formulated drug to animal models (e.g., mice, rats) via the target route (e.g., subcutaneous, oral).2. For biodistribution, use radiolabeled or fluorescently labeled compounds.3. Collect blood and tissue samples at set time points.	- Bioavailability: Determined from plasma concentration-time profiles (using PK parameters like AUC).- Tissue Distribution: Quantified by measuring radioactivity or fluorescence in dissected organs.

The Clinical Translation Pathway for IL Formulations

The journey from a promising preclinical result to an approved drug is structured and multi-staged. The translational science spectrum, as outlined by the NIH, provides a framework for this process [65]. The pathway for an IL-based therapeutic compound, from initial discovery to clinical trials, involves several critical stages.

Diagram 1: Clinical translation pathway for IL formulations.

Navigating the Preclinical to Clinical Transition

The transition from preclinical to clinical stages demands rigorous safety and manufacturing standardization [66].

• Preclinical In Vitro Evaluation: The first step involves confirming that the IL formulation does not alter the therapeutic's binding properties or mechanism of action through in vitro binding affinity and internalization assays [66].
• Preclinical In Vivo Models: Testing progresses to animal models to investigate target binding specificity, biodistribution, and pharmacokinetics, often in immunodeficient mice with induced tumor xenografts [66].
• Toxicity Studies: A critical gateway, this requires a single-dose, extended toxicity study in a single species to identify any adverse effects before human trials [66].
• Good Manufacturing Practice (GMP) Production: The therapeutic compound and its IL component must be manufactured under strict GMP conditions to ensure quality, purity, and consistency for human use [66].
• Regulatory Submission: All data is compiled into a comprehensive submission package, including the Investigational Medicinal Product Dossier (IMPD) and clinical trial protocol, for review by regulatory authorities before a first-in-human trial can begin [66].

The Scientist's Toolkit: Research Reagent Solutions

Successful research and development of IL formulations rely on a specific set of reagents, instruments, and methodologies.

Table 3: Essential Research Reagents and Tools for IL Formulation Development

Category	Item	Primary Function in IL Research
Biocompatible Ions	Choline Bicarbonate, L-Arginine, L-Histidine, Amino Acids	Serves as cations and anions for synthesizing low-toxicity, biocompatible ILs (e.g., Choline-Arginine).
Analytical Instruments	Rheometer, Size-Exclusion Chromatography (SEC-HPLC), Circular Dichroism (CD) Spectrometer, Gamma Counter	Measures critical quality attributes: viscosity, protein aggregation/truncation, secondary structure, and tissue biodistribution.
Model Systems	Cell Lines (e.g., for binding assays), Tumor Xenograft Mouse Models, Rat Intestinal Mucosae (ex vivo)	Provides platforms for evaluating binding specificity, in vivo efficacy/pharmacokinetics, and permeability enhancement.
Formulation Aids	Labrasol, Docusate (as anions), Polysorbates, Ethylcellulose (for spray-encapsulation)	Acts as permeation enhancers in API-ILs, stabilizers, or polymers for solidifying liquid IL formulations.

Current Challenges and Future Directions

Despite promising preclinical results, the clinical translation of IL formulations faces hurdles. A significant barrier has been the historical trade-off between using high IL concentrations for stability, which raises toxicity concerns, and using low IL concentrations, which limited the deliverable dose of the drug [63]. The development of ultra-high concentration antibody formulations (uHCAFs) using amino acid-based ILs like ChArg represents a pivotal advancement by overcoming this viscosity-toxicity hurdle [63]. Furthermore, the liquid nature of many ILs poses handling and dosage challenges, which are now being addressed through solidification techniques like spray-encapsulation with polymers such as ethylcellulose [64]. Future progress hinges on continued focus on designing ILs with inherently low toxicity and high biodegradability, scaling up GMP-compliant synthesis processes, and conducting rigorous long-term toxicology studies to satisfy regulatory requirements.

Ionic liquids (ILs) are a class of compounds defined as salts with a melting point below 100 °C, often liquid at room temperature [35] [67]. Their unique properties stem from their composition of bulky, asymmetric organic cations and smaller inorganic anions, which results in poor crystal packing and low melting points [35] [67]. A core characteristic of ILs is their tunability; the cation and anion can be selected or designed to impart specific physicochemical properties, making them "designer solvents" for a vast array of applications [35] [5]. This tunability is the foundational concept for their investigation in permeation enhancement, as their properties can be tailored to overcome specific biological barriers more effectively than traditional solvents.

The evolution of ILs is categorized into generations. First-generation ILs were primarily explored as green solvents, while second-generation ILs were engineered for specific applications like catalysis and electrochemistry [5]. The more advanced third-generation and fourth-generation ILs focus on bio-derived functionalities, sustainability, biodegradability, and multifunctionality, which are critical for biomedical applications [5]. A significant advancement within these later generations is the development of bioinspired ILs (BILs), which use cations like cholinium and guanidinium to improve biodegradability and reduce toxicity [35] [67].

Quantitative Performance Benchmarking

A critical comparison of ionic liquids against traditional chemical permeation enhancers and organic solvents reveals a distinct performance profile for ILs, characterized by their multifaceted mechanisms and high tailorability.

Table 1: Benchmarking Permeation Enhancers by Key Properties

Property	Ionic Liquids (ILs)	Traditional Chemical Permeation Enhancers (e.g., Azone, Ethanol, Fatty Acids)	Organic Solvents
Vapor Pressure	Very low to negligible [35] [67]	Variable (e.g., high for ethanol) [68]	Typically high [68]
Thermal Stability	High [35] [67]	Variable	Low to moderate
Tunability	Highly tunable via cation/anion selection [35] [5]	Low; fixed chemical structure	Low; fixed chemical structure
Primary Mechanism of Action	Disruption of lipid bilayers, protein denaturation, and lipid extraction [35] [67]	Fluidization of lipid bilayers (e.g., Azone, fatty acids) or solvent effects (e.g., ethanol) [69] [70]	Solvent effects and lipid extraction [68]
Typical Permeability Coefficient (Kp) Range	Wide range, highly tailorable	Data specific to each compound	~10-6 to ~10-3 cm/h (for neat solvents) [68]
Safety & Toxicity Profile	Variable; newer BILs are biodegradable and less toxic [35] [5]	Variable; can cause irritation and damage at effective concentrations [69]	Often high; requires careful handling [68]

Table 2: Experimental Permeation Data for Selected Organic Solvents [68]

Solvent	State	Lag Time (h)	Steady-State Flux (Jss, μmol/cm²/h)	Apparent Permeability Coefficient (Kp, cm/h)
Ethanol	Neat	0.4	28.90	1.70 x 10-3
Ethanol	Diluted (50%)	0.7	6.18	7.28 x 10-4
Methanol	Neat	0.3	34.20	2.02 x 10-3
Methanol	Diluted (50%)	0.3	14.60	1.72 x 10-3
Acetone	Neat	0.4	21.70	1.28 x 10-3
Acetone	Diluted (50%)	0.4	8.32	9.79 x 10-4
Dichloromethane	Neat	0.2	83.10	4.89 x 10-3

Mechanisms of Action: A Comparative Analysis

How Ionic Liquids Enhance Permeation

Ionic liquids enhance permeation through several sophisticated, and often synergistic, mechanisms that target the structure of the stratum corneum:

• Lipid Bilayer Disruption and Fluidization: The amphiphilic nature of ILs allows them to integrate into the lipid domains of the stratum corneum. The cations, particularly with longer alkyl chains, disrupt the highly ordered lipid bilayers, increasing their fluidity and creating diffusional pathways [35] [67]. This is analogous to the action of surfactants and fatty acids but is often more pronounced due to the ionic interactions.
• Extraction of Lipid Components: Certain ILs can actively dissolve and extract key lipid components from the stratum corneum, directly compromising the barrier's integrity [35].
• Interaction with Keratin: ILs can interact with and denature the intracellular keratin filaments in corneocytes, further reducing the diffusive resistance of the skin [35].
• Formation of Aqueous Pores: Some ILs have been shown to create aqueous pores in the skin structure, providing a route for hydrophilic molecules to permeate [67].

The structure of the IL directly influences its mechanism. For instance, increasing the alkyl chain length on the cation enhances its surfactant-like properties and its ability to disrupt lipids, but it may also increase toxicity [35].

Mechanisms of Traditional Enhancers and Solvents

• Traditional Chemical Permeation Enhancers (CPEs): Compounds like Azone (laurocapram) primarily work by fluidizing the stratum corneum lipids [70]. Fatty acids (e.g., oleic acid) create fluid domains within the lipid bilayers, while solvents like ethanol extract lipids and disrupt their packing, similarly to some ILs but often with less control [69] [70].
• Organic Solvents: Their action is primarily based on solvation and extraction. They can delipidize the stratum corneum, effectively removing the barrier, which can lead to high permeation but also significant irritation and damage [68]. Their high volatility can also make their action transient and difficult to control.

Diagram 1: Comparative mechanisms of permeation enhancers targeting the stratum corneum.

Experimental Protocols for Permeation Assessment

Standard In Vitro Skin Permeation Protocol

This protocol is widely used for evaluating permeation enhancers, as referenced in the studies on organic solvents and ILs [68] [35].

• 1. Skin Preparation: Use excised skin membranes. Porcine skin is a preferred model due to its similarity to human skin in terms of stratum corneum and epidermal thickness and permeability [68]. Dermatomed skin (200-400 μm thick) should be stored frozen and thawed before use.
• 2. Diffusion Cell Assembly: Use static Franz-type diffusion cells. The prepared skin membrane is mounted between the donor and receptor compartments, with the stratum corneum facing the donor side. The receptor chamber is filled with a suitable buffer (e.g., phosphate-buffered saline, PBS) and maintained at a constant temperature (e.g., 32°C) via a water jacket to mimic skin surface temperature [68].
• 3. Application of Test Formulation: The test formulation (containing the drug and the enhancer—IL, traditional CPE, or organic solvent) is applied to the donor compartment. For solvents, both neat and diluted forms are tested [68].
• 4. Sampling: At predetermined time intervals, aliquots (e.g., 300 μL) are withdrawn from the receptor compartment. The receptor volume is immediately replenished with fresh pre-warmed buffer to maintain sink conditions.
• 5. Analytical Quantification: The concentration of the permeated drug in the receptor samples is quantified using a validated analytical method, typically high-performance liquid chromatography (HPLC) or gas chromatography (GC) [68].
• 6. Data Analysis: Cumulative drug permeation is plotted against time. The steady-state flux (Jss, μg/cm²/h) is calculated from the slope of the linear portion. The permeability coefficient (Kp, cm/h) is calculated as Kp = Jss / Cd, where Cd is the donor concentration. The lag time (tlag, h) is determined from the x-intercept of the steady-state slope.

Diagram 2: Standard workflow for in vitro skin permeation assessment.

Protocol for Investigating IL-Skin Interactions

To understand the mechanism of IL action, additional techniques are employed alongside the permeation study [35]:

• Fourier-Transform Infrared Spectroscopy (FTIR): Used to detect changes in the lipid and protein structures of the stratum corneum. A shift in the symmetric and asymmetric CH₂ stretching absorption peaks indicates lipid bilayer fluidization. Changes in the Amide I and II bands reveal protein denaturation.
• Differential Scanning Calorimetry (DSC): Measures the thermal phase behavior of skin lipids after treatment with ILs. A decrease or disappearance of the endothermic peaks associated with lipid melting suggests that the IL has disrupted the highly ordered lipid structure.
• Confocal Microscopy: Utilizes fluorescent dyes to visualize the integrity of the skin and the pathways of drug penetration in the presence of ILs.

The Scientist's Toolkit: Key Research Reagents & Materials

Table 3: Essential Materials for Permeation Enhancement Research

Item / Reagent	Function & Application in Research	Example from Literature
Franz Diffusion Cell	The standard apparatus for in vitro permeation studies. Consists of donor and receptor chambers separated by a skin membrane.	Used to test percutaneous absorption of 38 organic solvents [68].
Excised Porcine Skin	A common and reliable model for human skin due to comparable permeability and structure.	Used as a model membrane in permeation studies of solvents and ILs [68] [35].
Room-Temperature ILs (RTILs)	The most common type of ILs studied for permeation enhancement, e.g., based on imidazolium, pyridinium, or cholinium cations.	1-butyl-3-methylimidazolium hexafluorophosphate ([BMIM][PF₆]) used in microemulsions [67].
Bioinspired ILs (BILs)	Newer-generation ILs designed for better biodegradability and lower toxicity, using cations like cholinium.	Cholinium-based ILs are highlighted as promising, safer alternatives [35].
Azone (Laurocapram)	A classic, "designer" chemical permeation enhancer used as a benchmark for lipid fluidization mechanisms.	Studied for buccal and transdermal delivery of drugs like propranolol and 5-fluorouracil [70].
Ethanol	A widely used organic solvent and penetration enhancer; acts by lipid extraction and fluidization.	Included in the panel of 38 tested organic solvents; data available for neat and diluted forms [68].
Attenuated Total Reflectance-FTIR (ATR-FTIR)	A spectroscopic technique to analyze the molecular-level interactions of enhancers with the skin in real-time.	Recommended for studying lipid fluidization and protein denaturation in the stratum corneum [35] [69].

The benchmarking analysis clearly establishes ionic liquids not merely as alternative solvents, but as a versatile and technologically advanced platform for permeation enhancement. Their key advantage lies in their inherent tunability, which allows researchers to design cations and anions that optimize permeation efficacy while managing toxicity—a level of control absent in traditional enhancers and organic solvents. While traditional solvents like dichloromethane can achieve high flux, they do so through non-specific, often damaging mechanisms and present significant safety risks [68]. In contrast, ILs, particularly third- and fourth-generation bioinspired ILs, offer a pathway to achieving high enhancement factors with a more favorable safety and environmental profile [35] [5].

The future of ILs in this field is intertwined with advancements in sustainability and smart design. The ongoing development of biodegradable ILs (BILs) will address critical environmental and toxicity concerns [35] [5]. Furthermore, the integration of artificial intelligence and computational modeling for predicting the physicochemical properties and biological interactions of novel IL structures will dramatically accelerate the discovery of bespoke enhancers for specific therapeutic agents [71]. As the market for ILs continues to grow, projected to reach USD 136.18 million by 2034, the driver for innovation in their biomedical applications will only intensify [71]. Ultimately, the transition from traditional solvents to tunable ILs represents a paradigm shift towards more precise, effective, and safer strategies for transdermal drug delivery.

Surface and interfacial properties, particularly wettability and oleophobicity, have become a critical area of research in material science and engineering. Wettability describes the ability of a liquid to spread on or repel from a surface, a property determined by the complex interplay between a surface's chemical composition and its micro- and nanoscale topography [72]. These properties hold particular significance for applications requiring extreme control of liquid interaction, including self-cleaning, anti-icing, dropwise condensation, anti-fogging, and enhanced fluid transport [72]. Within this domain, ionic liquids (ILs)—salts with low melting points and negligible vapor pressure—have emerged as particularly intriguing materials due to their tunable properties and unique interfacial behavior [73] [74].

This technical guide explores the fundamental principles, fabrication methodologies, and specialized applications of surfaces with engineered wettability and oleophobic characteristics, framed within the broader context of tunable ionic liquid research. The ability to precisely control these surface properties is opening new frontiers across biomedical engineering, energy systems, microfluidics, and tribology [75] [74].

Theoretical Foundations of Wettability

Fundamental Wettability Theories

The theoretical understanding of wettability begins with Young's equation, proposed in 1805, which describes the contact angle (θγ) formed at the solid-liquid-vapor interface on an ideal, smooth, homogeneous surface through a balance of interfacial tensions [75]:

cosθγ = (γsv - γsl)/γlv

where γsv, γsl, and γlv represent the solid-vapor, solid-liquid, and liquid-vapor interfacial tensions, respectively [75]. This equation forms the fundamental basis for wettability science, though it applies only to ideal surfaces.

For real-world, rough surfaces, two primary models extend Young's equation:

• Wenzel Model: Accounts for surface roughness (r) by assuming complete liquid penetration into surface textures, which amplifies the intrinsic wettability: cosθ* = rcosθY [72] [75].
• Cassie-Baxter Model: Describes a composite interface where liquid bridges across surface roughness, trapping air pockets: cosθCB = fscosθs + facosθa [72] [75].

For underwater oleophobicity, a modified Cassie-Baxter equation applies where surface grooves are water-filled rather than air-filled [76]:

cosθCBoil = f'scosθ's + fwcosθw

Advanced Wettability States and Liquid Adhesion

Beyond basic hydrophobicity and hydrophilicity, surfaces can exhibit extreme "super" states including superhydrophilicity (contact angle θ < 10°), superhydrophobicity (θ > 150°), superoleophilicity, and superoleophobicity [72] [75] [77]. These superwetting states demonstrate particular importance in biomedical applications, where they significantly influence protein adsorption, bacterial adhesion, and cell attachment [75].

Liquid adhesion on surfaces is quantified through several parameters:

• Contact Angle Hysteresis (CAH): The difference between advancing (θAdv) and receding (θRec) contact angles [76].
• Roll-off Angle (θR): The minimum tilt angle required to make a droplet roll off the surface [76].
• Adhesion Force: Directly measured using microelectromechanical balances for microliter-sized droplets [76].

Based on wettability and adhesion characteristics, liquid behavior on surfaces can be classified into four distinct contact states [77]:

Table 1: Liquid Contact States on Solid Surfaces

Contact State	Description	Liquid Behavior	Natural Example
Lyophilic	Ultra-high adhesion	Droplet wets or spreads flat on surface	-
Pinning	Lyophobic with high adhesion	Spherical droplet that stays pinned on surface	Rose petal
Slippery	Lyophobic with low adhesion	Spherical droplet that easily slides/rolls off	Lotus leaf
Anisotropic	Direction-dependent adhesion	Droplet moves preferentially in one direction	Rice leaf

Figure 1: Theoretical Evolution of Wettability Models

Fabrication Strategies for Tunable Wettability

Surface Texturing and Coating Techniques

Advanced fabrication methods enable precise control over surface topography and chemistry to achieve desired wettability states. Laser surface texturing with ultrafast lasers has emerged as a versatile approach for creating micro- and nanoscale surface features with minimal heat-affected zones [72]. When combined with plasma-enhanced chemical vapor deposition (PECVD) coatings, this "LasPlas" process creates surfaces with tunable, permanent, and instantly available super-wettability states [72].

Key laser texturing parameters include:

• Pulse Duration: Femtosecond to picosecond pulses (e.g., τ = 260 fs)
• Repetition Rate: Typically 200 kHz
• Scanning Speed and Hatching Distance: Controls texture density and pattern
• Texturing Rate: Defined as Ȧ = (vx · py)/n where vx is scanning speed, py is line hatching distance, and n is number of scans [72]

PECVD coatings applied to textured surfaces include:

• Silicone-like Polymer Coatings: Using HMDSO precursor for hydrophobic properties
• PTFE Coatings: Using C4F8 precursor for superhydrophobic surfaces
• Glass (SiOx) Coatings: Using HMDSO with oxygen process gas for superhydrophilic surfaces [72]

Bioinspired and Template-Based Approaches

Natural organisms provide sophisticated blueprints for designing oil-repellent surfaces. Fish scales exhibit underwater superoleophobicity due to their hydrophilic calcium phosphate skeleton, mucus coating, and rough micro/nano surface texture [76]. Similarly, shark skin with ribbed, hydrophilic scales and a mucus layer demonstrates oil-repellent properties that prevent biofouling [76].

Common bioinspired fabrication techniques include:

• Template Methods: Using natural or synthetic templates (e.g., porous anodic aluminum oxide) to replicate specific surface patterns [76]
• Hydrothermal Methods: Employing elevated temperatures and pressures to grow hierarchical structures like TiO2 flowers [76]
• Electrospinning: Creating micro/nanostructured fibers with controlled wettability [76]

Table 2: Fabrication Techniques for Surfaces with Specific Wettability

Fabrication Method	Key Features	Resulting Wettability	Limitations
Laser Texturing + PECVD	Permanent, tunable, instant super-wettability	Superhydrophilic (θ < 10°) or superhydrophobic (θ > 150°)	Requires specialized equipment
Template Method	Direct adoption of natural patterns	Underwater superoleophobicity	Template removal may damage structures
Hydrothermal Method	High-purity, uniform roughness	Superhydrophilicity and underwater superoleophobicity	Challenging to scale up
Electrospinning	Micro/nanostructured fibers	Tunable oleophobicity/hydrophobicity	Limited to fiber-based substrates

Ionic Liquids at Interfaces

Unique Interfacial Properties of Ionic Liquids

Ionic liquids exhibit distinctive interfacial behavior due to their complex molecular structures and ability to self-assemble at interfaces. Unlike simpler organic liquids, ILs demonstrate surface enrichment of specific molecular moieties according to the "Langmuir principle," where components with the least attractive interactions in the bulk preferentially populate the outer surface [73]. This results in unique interfacial compositions that determine their surface tension and wettability characteristics.

Molecular dynamics simulations of [bmim][triflate] reveal that the alkyl chains of [bmim] cations orient toward the vacuum interface, creating an organized interfacial layer with the triflate anions positioned deeper in the liquid [78]. This molecular arrangement significantly influences the surface tension, which shows linear temperature dependence across a wide range (323.15 to 573.15 K) [78].

The surface tension of ILs is highly sensitive to environmental conditions. For [C8C1Im][PF6], surface tension measured under high vacuum is consistently higher than under ambient conditions due to the absence of water uptake, highlighting the importance of ultraclean measurement conditions for obtaining intrinsic property values [73].

Tribological Behavior and Lubrication Mechanisms

Ionic liquids demonstrate remarkable tribological properties due to their novel self-assembling behavior at solid-liquid interfaces, where they remain firmly surface-adsorbed under high normal and shear stress [74]. This makes them particularly valuable as lubricants in demanding applications.

The lubrication mechanisms of ILs involve:

• Interfacial Layering: Formation of ordered molecular layers near solid surfaces
• Tribolayer Formation: Chemical reaction layers under high pressure and temperature
• Molecular Structuring: Dependence on substrate surface chemistry and topography [74]

The composition of confined thin IL films varies with surface properties, environmental conditions, and the chemical structure of the ionic liquid itself, making generalizations challenging and highlighting the need for tailored IL selection for specific tribological applications [74].

Experimental Methodologies and Characterization

Surface Tension and Contact Angle Measurement

Accurate characterization of surface and interfacial properties requires specialized experimental approaches under controlled conditions:

Pendant Drop Method: This technique determines surface tension from the shape of a hanging drop deformed by gravity. The method is based on the Bond number (β = ΔρgR₀²/γ), which represents the ratio of gravitational to interfacial forces [73]. Advanced axisymmetric drop shape analysis (ADSA) algorithms are used to calculate surface tension from drop morphology [73].

Sessile Drop Method: For solid-liquid interfacial tension measurements, the contact angle of a sessile drop is analyzed using Young's equation: cosθ = (γSG - γSL)/γLG [73]. This approach requires ultraclean conditions to obtain intrinsic interfacial properties unaffected by surface contaminants.

High-Vacuum Measurements: Novel experimental setups now enable surface tension and contact angle measurements under high vacuum conditions (10⁻⁷ mbar), eliminating the confounding effects of atmospheric contaminants and providing benchmark data for theoretical calculations [73].

Figure 2: Experimental Workflow for Surface Engineering

Research Reagent Solutions and Materials

Table 3: Essential Research Reagents and Materials for Wettability Studies

Material/Reagent	Chemical Composition	Function in Research	Application Examples
Ionic Liquid [bmim][triflate]	1-Butyl-3-methylimidazolium trifluoromethanesulfonate	Model ionic liquid for interfacial studies	Surface tension benchmarks, molecular dynamics validation [78]
Ionic Liquid [C8C1Im][PF6]	1-Octyl-3-methylimidazolium hexafluorophosphate	Surface-active ionic liquid	Surface tension studies in vacuum vs. air [73]
HMDSO Precursor	Hexamethyldisiloxane (C6H18OSi2)	PECVD precursor for silicone-like polymer or glass coatings	Hydrophobic (polymer) or superhydrophilic (SiOx) coatings [72]
C4F8 Precursor	Octafluorocyclobutane	PECVD precursor for PTFE-like coatings	Superhydrophobic and oleophobic surfaces [72]
AISI 304 Stainless Steel	Fe/Cr/Ni alloy	Metallic substrate for texturing and coating	Industrial applications requiring corrosion resistance [72]

Applications in Biomedical Engineering and Specialized Fields

Biomedical Applications

Engineered wettability has found significant applications in biomedical engineering, where surface properties directly influence biological interactions:

• Tissue Engineering: Superhydrophobic-superhydrophilic patterned surfaces enable precise micropatterning of living cells for tissue constructs and cell-based microarrays [75]
• Anti-Biofouling: Superhydrophobic surfaces effectively resist bacterial adhesion, protein adsorption, and blood coagulation, making them ideal for implant materials [75]
• Biosensing: Wettability-patterned surfaces facilitate controlled biomolecular deposition for enhanced sensor sensitivity and specificity [75]
• Medical Devices: Slippery liquid-infused porous surfaces (SLIPS) exhibit exceptional repellency to physiological fluids, proteins, and cells, making them suitable for medical tubing and implants [75]

Stimuli-Responsive and Smart Surfaces

Surfaces with dynamically tunable wettability represent an advanced frontier in material science. These smart surfaces can reversibly switch their wettability and adhesion characteristics in response to external stimuli [77]:

• Stretching-Responsive Surfaces: Mechanical strain alters surface geometry and roughness, enabling switching between pinning and slippery states [77]
• Photo-Responsive Surfaces: Light-induced molecular changes modify surface energy and wettability
• Electro-Responsive Surfaces: Applied electrical potentials reorganize surface molecules or induce electrowetting
• Thermo-Responsive Surfaces: Temperature-sensitive polymers transition between expanded and collapsed states
• pH-Responsive Surfaces: Chemical groups protonate/deprotonate to alter surface charge and wettability [77]

These stimuli-responsive materials enable advanced applications in microfluidics, droplet manipulation, liquid transport, and flexible smart devices [77].

Energy and Industrial Applications

Specialized wettability surfaces play crucial roles in energy systems and industrial processes:

• Enhanced Heat Transfer: Superhydrophobic surfaces promote dropwise condensation for improved cooling efficiency in thermal management systems [72]
• Corrosion Resistance: Superhydrophobic coatings on metals provide barrier protection against corrosive liquids [72]
• Oil/Water Separation: Underwater superoleophobic membranes enable efficient separation of oil-water mixtures for environmental remediation [76]
• Tribological Applications: Ionic liquids serve as high-performance lubricants forming protective boundary layers on contacting surfaces [74]

The engineering of surface and interfacial properties with unique wettability and oleophobic characteristics represents a rapidly advancing field with significant implications across biomedical, energy, and industrial domains. The integration of advanced fabrication techniques like laser texturing and PECVD coating with the unique properties of ionic liquids enables unprecedented control over liquid-surface interactions. As characterization methods become more sophisticated and our understanding of interfacial phenomena deepens, the design of next-generation functional surfaces with precisely tailored wettability will continue to expand the boundaries of materials science and engineering.

The future of this field lies in developing multifunctional, responsive surfaces that adapt to changing environmental conditions, self-heal from damage, and integrate multiple wettability states for complex liquid manipulation tasks. Such advances will further unlock the potential of tailored surface properties to address challenges in healthcare, energy sustainability, and advanced manufacturing.

Conclusion

Ionic liquids represent a paradigm shift in pharmaceutical technology, offering a uniquely tunable platform to simultaneously address the critical challenges of poor drug solubility, instability, and inadequate targeting. Their modular nature allows for precise customization of properties to meet specific therapeutic needs, from stabilizing delicate biologics to enabling non-invasive transdermal delivery of macromolecules. As research progresses, the future of ILs lies in the development of smarter, biodegradable formulations, accelerated by AI-driven design and advanced manufacturing techniques like 3D printing. With several IL-based formulations already advancing through clinical trials, these versatile materials are poised to transition from laboratory innovations to mainstream clinical solutions, ultimately enabling more effective, patient-friendly medicines and redefining the landscape of drug delivery and development.

References

• 1. Molecule(s) of Interest: I. Ionic Liquids–Gateway to Newer ... [https://www.mdpi.com/1422-0067/23/22/14346]
• 2. Ionic Liquids in Pharmaceutical and Biomedical Applications [https://www.mdpi.com/1999-4923/16/1/151]
• 3. Revisiting Ionic Liquid Structure-Property Relationship [https://pmc.ncbi.nlm.nih.gov/articles/PMC7589445/]
• 4. Ionic Liquids: Extraordinary properties and potential for ... [https://cicenergigune.com/en/blog/ionic-liquids-extraordinary-properties-and-potential-for-clean-hydrogen-generation-thermal-energy-storage]
• 5. A review on the evolution of ionic liquids: Sustainable ... [https://www.sciencedirect.com/science/article/pii/S2589234725000892]
• 6. Ionic Liquids in Analytical Chemistry [https://pmc.ncbi.nlm.nih.gov/articles/PMC11912132/]
• 7. An Overview on the Role of Ionic Liquids and Deep ... [https://pmc.ncbi.nlm.nih.gov/articles/PMC11946670/]
• 8. Ionic liquids and their bases: Striking differences in the ... [https://www.nature.com/articles/srep16876]
• 9. The quantitative structure-property relationships for the gas ... [https://www.sciencedirect.com/science/article/abs/pii/S0167732221022984]
• 10. Ion conformation and orientational order in a dicationic ... [https://www.nature.com/articles/s41598-021-85021-y]
• 11. Modular Design via Multiple Anion Chemistry of the High ... [https://pmc.ncbi.nlm.nih.gov/articles/PMC7007234/]
• 12. Orthogonal, modular anion–cation and cation–anion self ... [https://pmc.ncbi.nlm.nih.gov/articles/PMC9993851/]
• 13. Experiences with an Inquiry-Based Ionic Liquid Module in ... [https://pmc.ncbi.nlm.nih.gov/articles/PMC11097389/]
• 14. Ionic liquid [https://en.wikipedia.org/wiki/Ionic_liquid]
• 15. Ionic Liquids in Pharmaceutical and Biomedical Applications [https://pmc.ncbi.nlm.nih.gov/articles/PMC10818567/]
• 16. Emerging applicative areas of ionic liquids in biomedical ... [https://www.sciencedirect.com/science/article/abs/pii/S016773222500621X]
• 17. A review on the evolution of ionic liquids: Sustainable ... [https://ui.adsabs.harvard.edu/abs/2025MTSus..3101160A/abstract]
• 18. Ionic liquids: a brief history - PMC [https://pmc.ncbi.nlm.nih.gov/articles/PMC5988633/]
• 19. Recent Advances in Biocompatible Ionic Liquids in Drug ... [https://www.mdpi.com/1999-4923/15/4/1179]
• 20. Exploring the Biological Activities of Ionic Liquids and Their ... [https://www.mdpi.com/2076-393X/13/4/365]
• 21. A Comprehensive Review of the Thermophysical ... [https://www.mdpi.com/1996-1073/18/2/267]
• 22. Cholinium amino acid-based ionic liquids - PMC [https://pmc.ncbi.nlm.nih.gov/articles/PMC7930144/]
• 23. The Role of Ionic Liquids in the Pharmaceutical Field [https://www.mdpi.com/1422-0067/21/21/8298]
• 24. Ionic liquids revolutionizing drug delivery [https://www.sciencedirect.com/science/article/abs/pii/S0168365925009691]
• 25. Active Pharmaceutical Ingredient-Ionic Liquids (API-ILs) [https://www.mdpi.com/1420-3049/27/16/5117]
• 26. Exploring the Biological Activities of Ionic Liquids and Their ... [https://pmc.ncbi.nlm.nih.gov/articles/PMC12031513/]
• 27. Ionic Liquids: Transforming Sustainability in the U.S. Market [https://roco.global/ionic-liquids-us-market-sustainable-innovation/?srsltid=AfmBOoqaNWXBUcw0FIFPiaJghQniO_NCIZNLQBrAiuHOWYUZC-g7V-Tc]
• 28. Ionic liquids self-assembled micelles for transdermal delivery [https://www.sciencedirect.com/science/article/pii/S2590156725000350]
• 29. Ionic liquids self-assembled micelles for transdermal delivery [https://pubmed.ncbi.nlm.nih.gov/40688029/]
• 30. Unveiling the interaction between micelles of different ... [https://www.sciencedirect.com/science/article/abs/pii/S2468519425001521]
• 31. Synthesis and physical properties of tunable aryl alkyl ionic ... [https://www.beilstein-journals.org/bjoc/articles/20/110]
• 32. Cubosomes from hierarchical self-assembly of poly(ionic ... [https://www.nature.com/articles/ncomms14057]
• 33. Cationic alkyl chain length and nanoaggregate form of ... [https://www.nature.com/articles/s41467-025-62206-x]
• 34. Role and Recent Advancements of Ionic Liquids in Drug ... [https://pmc.ncbi.nlm.nih.gov/articles/PMC9963759/]
• 35. Ionic Liquids as Potential and Synergistic Permeation ... [https://pmc.ncbi.nlm.nih.gov/articles/PMC6409523/]
• 36. Treatment of psoriasis with NFKBIZ siRNA using topical ... [https://pmc.ncbi.nlm.nih.gov/articles/PMC7439648/]
• 37. Engineered choline–Salcaprozate ionic liquids for ... [https://www.sciencedirect.com/science/article/abs/pii/S0021979725028553]
• 38. Permeation Enhancer‐based Ionogel Shows Remarkable ... [https://pmc.ncbi.nlm.nih.gov/articles/PMC12333474/]
• 39. Ionic Liquid Market Size, Trends, Share & Forecast Report ... [https://www.mordorintelligence.com/industry-reports/ionic-liquid-market]
• 40. Transdermal delivery of Fn14 siRNA using a novel ... [https://www.sciencedirect.com/science/article/abs/pii/S0168365923007939]
• 41. Stabilization challenges and aggregation in protein-based ... [https://pmc.ncbi.nlm.nih.gov/articles/PMC10711991/]
• 42. Biopharmaceutical Product Stability Considerations, Part 1 [https://www.bioprocessintl.com/formulation/stability-considerations-for-biopharmaceuticals-overview-of-protein-and-peptide-degradation-pathways]
• 43. Development of a Technique for Quantifying Protein ... [https://www.biopharminternational.com/view/development-technique-quantifying-protein-degradation]
• 44. Targeted Protein Degradation with PROTACs and ... [https://blog.crownbio.com/targeted-protein-degradation-with-protacs-and-molecular-glues]
• 45. Ionic liquids for advanced materials [https://www.sciencedirect.com/science/article/abs/pii/S2588842021000511]
• 46. Transdermal Drug Delivery Systems: Methods for Enhancing ... [https://pmc.ncbi.nlm.nih.gov/articles/PMC12300492/]
• 47. An overview of biomedical applications of choline geranate ... [https://pmc.ncbi.nlm.nih.gov/articles/PMC9468656/]
• 48. Transdermal Permeation Assays of Curcumin Aided by ... [https://www.mdpi.com/1999-4923/14/4/779]
• 49. Design Principles of Ionic Liquids for Transdermal Drug ... [https://pubmed.ncbi.nlm.nih.gov/31112631/]
• 50. Advancing Ionic Liquid Research with pSCNN: A Novel ... [https://pmc.ncbi.nlm.nih.gov/articles/PMC11270577/]
• 51. Ionic Liquids in Analytical Chemistry: Fundamentals, Technological Advances, and Future Outlook - Consensus [https://www.consensus.app/papers/details/b237df8957325ae59ecae8ecd51c1337/]
• 52. the expanding role of ionic liquids in dermatological drug ... [https://www.sciencedirect.com/science/article/abs/pii/S0378517325009974]
• 53. Sustainable approach for synthesis of new coumarin-linked ... [https://www.nature.com/articles/s41598-025-97949-6]
• 54. An ionic liquid nanoemulsion transdermal delivery system ... [https://www.sciencedirect.com/science/article/abs/pii/S0168365925008557]
• 55. An ionic liquid nanoemulsion transdermal delivery system ... [https://pubmed.ncbi.nlm.nih.gov/40975175/]
• 56. Fungal infections: New challenges in treatment [https://www.cas.org/resources/cas-insights/treating-fungal-infections]
• 57. Antifungal Efficacy of Plasma-Activated Liquids against ... [https://pmc.ncbi.nlm.nih.gov/articles/PMC12612981/]
• 58. Design Principles and Applications of Ionic Liquids for ... [https://pmc.ncbi.nlm.nih.gov/articles/PMC11578336/]
• 59. Theoretical models of solubility for organic solvents. A ... [https://www.sciencedirect.com/science/article/abs/pii/S016773222400792X]
• 60. Machine learning-driven generation and screening of ... [https://jcheminf.biomedcentral.com/articles/10.1186/s13321-025-01018-z]
• 61. Simulating the ionic liquid mixing with organic-solvent ... [https://www.nature.com/articles/s41598-024-74561-8]
• 62. Recent Advances in Biocompatible Ionic Liquids in Drug ... [https://pmc.ncbi.nlm.nih.gov/articles/PMC10145603/]
• 63. Ultra-high concentration low-viscosity subcutaneous ... [https://www.sciencedirect.com/science/article/abs/pii/S0168365925009083]
• 64. Solidified ionic liquid-based formulations of metformin with ... [https://pubmed.ncbi.nlm.nih.gov/40320021/]
• 65. Translational Science Spectrum [http://ncats.nih.gov/about/about-translational-science/spectrum]
• 66. Clinical Translation of Your Compound in 6 Steps | TRACER [https://www.tracercro.com/resources/blogs/fast-clinical-translation-of-your-therapeutic-compound-a-stepwise-approach/]
• 67. Ionic Liquids as Potential and Synergistic Permeation ... [https://www.mdpi.com/1999-4923/11/2/96]
• 68. Percutaneous absorption of thirty-eight organic solvents in ... [https://pmc.ncbi.nlm.nih.gov/articles/PMC6209206/]
• 69. Skin penetration enhancers: Mechanistic understanding ... [https://pubmed.ncbi.nlm.nih.gov/39982640/]
• 70. Review Mechanisms of oral permeation enhancement [https://www.sciencedirect.com/science/article/abs/pii/S0378517397001932]
• 71. Ionic Liquids Market Size to Hit USD 136.18 Million by 2034 [https://www.precedenceresearch.com/ionic-liquids-market]
• 72. Tunable, permanent and instantly available super ... [https://www.nature.com/articles/s41598-025-11750-z]
• 73. Probing Surface and Interfacial Tension of Ionic Liquids in ... [https://www.mdpi.com/1422-0067/23/21/13158]
• 74. Ionic liquids at interfaces and their tribological behavior [https://experts.illinois.edu/en/publications/ionic-liquids-at-interfaces-and-their-tribological-behavior-2]
• 75. Tailoring Materials with Specific Wettability in Biomedical ... [https://pmc.ncbi.nlm.nih.gov/articles/PMC8498887/]
• 76. Strategies to modulate underwater oil wettability and ... [https://www.sciencedirect.com/science/article/abs/pii/S0001868625000533]
• 77. Stimuli-responsive surfaces for switchable wettability and ... [https://pmc.ncbi.nlm.nih.gov/articles/PMC8205534/]
• 78. Interfacial properties of the ionic liquid [bmim][triflate] over a ... [https://pmc.ncbi.nlm.nih.gov/articles/PMC9078723/]