Development, Characterization, and Evaluation of CAGE-based Ionic Liquid Systems for Transdermal Delivery

Summary

This protocol discusses standardization for the preparation of CAGE-based ionic liquid systems to ensure reproducibility and streamline the overall workflow across different scientific labs.

Abstract

Ionic liquids have recently gained remarkable recognition for their applications within drug delivery, in particular their potential to enable cutaneous drug penetration and enhance drug permeation. In addition to enhancing barrier permeability, ionic liquids have excellent solvation properties, addressing the challenge of dissolving poorly soluble drugs. Due to the large asymmetry between the organic cations and anions, these functional salts remain liquid at room temperature. From an application perspective, ionic liquids provide a promising and highly tunable approach to serve multiple therapeutic areas. A biocompatible ionic liquid composed of choline and geranic acid (CAGE), in a stoichiometric ion ratio of 1:2, holds immense potential for transdermal delivery of a wide range of active pharmaceutical ingredients, including small molecules, peptides, proteins, and nucleic acids. This has a minimal impact on skin barrier function. Herein, we share our methods for the preparation, characterization, and evaluation of CAGE. Representative results are shown for CAGE characterization using NMR spectroscopy, differential scanning calorimetry, Karl-Fischer titration for water content analysis, and ex vivo barrier interaction studies on porcine skin.

Introduction

The skin is the body's largest and most vital organ, serving as a protective barrier against harmful substances while also preventing water loss¹. At the same time, applying drugs to and through the skin offers a wide range of advantages. Compared to injectables and oral delivery, cutaneous administration increases patient compliance and avoids first-pass metabolism in the liver². Especially for skin conditions, cutaneously administered formulations excel in providing a localized effect, diminishing the systemic exposure³. However, the almost impermeable nature of the skin significantly challenges....

Protocol

All experimental procedures were carried out in compliance with the European Union Directive 2010/63 and were approved by the Danish Veterinary and Food Administration (License No. DK-13-oth-931833).

1. CAGE preparation by salt metathesis reaction

1. Recrystallization of geranic acid (85% purity, mol wt 168.23 g/mol)
   1. Take a 500 mL round-bottom flask and measure its weight.
   2. Weigh 40.0 g geranic acid in the flask. This corresponds to 0.2 mol of geranic acid.
   3. Recrystallize geranic acid in a 70:30% (w/w) geranic acid:acetone: Add (40 g*0.85*0.30)/(0.70*0.784 g/mL) = 18.42 mL of ac....

Results

NMR-spectra
The goal of this technique is to confirm the successful formation of CAGE by analyzing the chemical environments of hydrogen and carbon atoms using ¹H and ¹³C NMR spectroscopy. The resulting spectra provide characteristic chemical shifts, splitting patterns, and integration values that reflect the molecular structure and composition of the synthesized IL. The proton NMR spectrum in Figure 1 confirms the 2:1 molar ratio of geranic acid to.......

Discussion

The preparation method of CAGE described in this work was adopted from the literature¹⁵. However, it is important to acknowledge that the synthesis process is sensitive to various external factors, which can influence the properties of the final product. Therefore, once prepared, the CAGE system should be characterized using techniques such as NMR spectroscopy to confirm the formation of CAGE.

As shown in Figure 1, the proton spectra of CAG.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
5 mm NMR tubes	Sigma Aldrich	41121705
Acetone	VWR	23L064039
Aluminium Tzero Pans	TA Instruments	90,16,83,901
Aqua Flux Device	Biox Systems
Bruker Avance III NMR	Bruker		Instrument for NMR spectroscopy
Choline Bicarbonate	Sigma Aldrich	C7519
Dermatome	Zimmer Biomet
Differential Scanning Calorimeter	TA Instruments
Ethanol	VWR	23J314009
Expanded Polystyrene	Styrofoam
Franz Diffusion Cells	PermeGear
Geranic Acid	Sigma Aldrich	427764
Grafting Tape	Parafilm
Karl Fischer Titrator	Mettler Toledo	30252662
Methanol	VWR	85681.32
PBS	Fischer Scientific	10388739
TA universal analysis 2000 software	TA Instruments		Software for DSC data analysis
TopSpin Software	Bruker		Software for NMR data analysis