Biocompatible Ionic Liquids as Designer Solvents for Non-lamellar Lyotropic Liquid Crystalline Nanoparticles
Creators: name: El Mohamad, Mohamad
Publisher: name: RMIT University
Year: 2024
Subjects: classificationCode: 510405
subject: Soft condensed matter
classificationCode: 340603
subject: Colloid and surface chemistry

description: Nanomedicine stands as the foremost frontier in drug delivery formulations, and the interest in these systems is continuously increasing. Lipid nanoparticles are heavily investigated due to the capacity of lipids to self-assemble into a variety of structures with distinctive properties. The biocompatibility, cost-effectiveness, and ease of production further accentuate their appeal. Lipid-based lyotropic liquid crystalline nanoparticles (LCNPs) particularly stand out for their ordered structures and high surface to volume ratio, rendering them suitable for delivering a diverse range of therapeutics. However, like all nanoparticles, they confront challenges related to stability post-administration and interaction with blood components, which influence their biological fate. Moreover, these particles are characterised by a structure-function relationship, rendering their preparation by rational design necessary for specific applications. To address these challenges and enhance their function for targeted delivery, manipulating their structures and surface characteristics is often achieved by incorporating surfactants into the main lipid matrix. Ionic liquids (ILs), a versatile class of tailorable solvents composed of ions, emerge as promising solvent additives for this purpose. Biocompatible ILs have demonstrated potential for biological applications owing to their low cytotoxicity and high biodegradability. These ILs have shown remarkable efficacy in optimising nanoparticles, offering improved stability during production and enhanced functions. Moreover, a wide array of biocompatible ILs support the self-assembly of amphiphiles as solvents and co-solvents. Despite these advancements, there remains a gap in research regarding the exploration of biocompatible ILs as solvent additives in LCNP formulations. This gap formed the basis for this project, where the primary objective was to investigate how ILs can be harnessed as additives to modulate the nanostructure of LCNPs. Additionally, the project explored the potential of ILs as functional ingredients, particularly their ability to modify the surface characteristics of LCNPs and potentially enhance their efficacy in hydrophobic drug delivery.
First, a library of 13 choline-based ionic liquids (ILs), commonly described in the literature for biomedical applications, was synthesised. These included 10 choline amino acid ILs, in addition to choline acetate and choline geranate at 2 molar ratios. Their cytotoxicity was examined in 4 cell lines; Human dermal fibroblasts (HDF), epidermoid carcinoma cells (A431), cervical cancer cells (HeLa), and gastric cancer cells (AGS). Their hemolytic activity towards red blood cells was also studied. The results indicated that the toxicity and hemolytic properties of these ILs are mainly influenced by the anion’s hydrophobicity and the pH of the IL solutions. To investigate any correlation between the ILs cytotoxicity and their effects on cell membrane, quartz crystal microbalance with dissipation (QCM-D) and total internal reflection fluorescence (TIRF) microscopy were employed to clarify how selected ILs interact with model lipid membranes made of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC). The QCM-D data revealed that ILs with higher toxicity had more significant interactions with the membrane, leading to bigger changes in membrane fluidity and mass. Moreover, TIRF microscopy revealed that choline acetate, the least toxic IL, induced lipid rearrangements and pore formation in the membrane, while choline geranate, a hydrophobic IL amongst the most toxic, disrupted the lipid bilayer. These results highlight the correlation between the cytotoxicity and membrane interactions, and underline the importance of understanding the impact of ILs on cellular membranes in their biocompatibility assessment and design for biomedical applications.
The library of ILs was then explored as solvent additives for Monoolein (MO)-based LCNPs. Dynamic light scattering, small angle X-ray scattering, and cryogenic transmission electron microscopy were used to analyse the physical characteristics of 78 LCNP formulations prepared in ILs aqueous solutions, at six concentrations: 20, 10, 5, 1, 0.33, and 0.16 wt% in H2O. The results revealed the presence of particles with various nanostructures, including cubosomes, hexosomes, and micellar cubosomes. The internal nanostructures of the MO nanoparticles were influenced by the IL anion, IL concentration, but the ILs solution’s pH was the dominant factor. 
Phytantriol (PHY)-based LCNP formulations prepared in IL aqueous solutions provided a pH-independent system to investigate the effect of the structure of the IL components on the nanostructure of the particles. The ILs library was extended to 21 choline-based biocompatible ILs, with the addition of 8 choline carboxylic acid ILs composed of anions with various chain lengths. The particles were also incubated with human serum and serum proteins to examine the effect of ILs of the surface characteristics of the particles, and their interactions with the biological media. SAXS analysis revealed that the nanostructure of the LCNPs was dependent on both the concentration of ILs and the hydrophobicity of the anions. Upon incubation with human serum, a transition from the inverse bicontinuous cubic mesophase (Q2) to the inverse hexagonal (H2) mesophase occurred. The rate of this transition was influenced by the specific IL used. Liquid chromatography-mass spectrometry (LC-MS) and proteomics analysis of selected samples were used to identify the protein corona composition of the particles, which was dominated by albumin, apolipoproteins, and serotransferrin. Notably, the abundancy of these proteins in the protein corona varied among the samples. The results obtained from both the MO and PHY-based formulations illustrate the effectiveness of IL additives for tailoring the internal nanostructure of lipid-based LCNPs, and modifying their surface characteristics.
The incorporation of ILs into lipid cubosome formulations to enhance their effectiveness as nanocarriers for hydrophobic drug delivery was then explored. MO cubosomes control sample, and MO cubosomes containing the hydrophilic IL choline glutamate [Cho][Glu] at a 70:30 MO:[Cho][Glu] molar ratio, and the hydrophobic IL choline geranate [Cho][Ge] at a 98:2 MO:[Cho][Ge] molar ratio were prepared to encapsulate the hydrophobic drug paclitaxel (PTX). Our findings indicate that cubosomes with [Cho][Ge] maintained their small size and low size distribution post drug encapsulation. In contrast, both the MO control and MO:[Cho][Glu] samples showed an increase in size and size distribution after drug encapsulation. Additionally, the presence of ILs significantly boosted the PTX encapsulation efficiency of the cubosomes, increasing it from 35% in the MO control to 61% in the MO:[Cho][Glu] sample and 78% in the MO:[Cho][Ge] cubosomes. All cubosome formulations exhibited a two-stage drug release profile (burst and sustained). CellTiter Blue cell toxicity assays demonstrated that all cubosomes effectively inhibited cancer cells growth. 
The findings of this project highlight the effectiveness of employing IL additives as a strategy to enhance LCNPs effectiveness for drug delivery purposes.