Ionic Liquid-Based Immunization Patch for the Transdermal Delivery of Antigens.

Herein, we report a transdermal patch prepared using an ionic liquid-based solid in oil (IL-S/O) nanodispersion and a pressure-sensitive adhesive (PSA) to deliver the macromolecular antigenic protein, ovalbumin (OVA). The IL-S/O nanodispersion and a PSA were first mixed at an equal weight ratio, then coated onto a release liner, and covered with a support film. To evaluate the effect of the PSA, three types of PSAs, DURO-TAK 87-4098, DURO-TAK 87-4287, and DURO-TAK 87-235A, were used to obtain the corresponding IL-S/O patches SP-4098, SP-4287, and SP-235A, respectively. The prepared IL-S/O patches were characterized for surface morphology, viscoelasticity, and moisture content. In vitro skin penetration and in vivo immunization studies of the IL-S/O patches were performed using Yucatan micropig skin and the C57BL/6NJc1 mice model, respectively. The SP-4098 and SP-4287 delivered 5.49-fold and 5.47-fold higher amounts of drug compared with the aqueous formulation. Although both patches delivered a similar amount of drug, SP-4287 was not detached fully from the release liner after 30 days, indicating low stability. Mice immunized with the OVA-containing SP-4098 produced a 10-fold increase in anti-OVA IgG compared with those treated with an aqueous formulation. These findings suggested that the IL-S/O patch may be a good platform for the transdermal delivery of antigen molecules.

Design and validation of functionalized redox-responsive hydrogel beads for high-throughput screening of antibody-secreting mammalian cells.

Antibody drugs play a vital role in diagnostics and therapy. However, producing antibodies from mammalian cells is challenging owing to cellular heterogeneity, which can be addressed by applying droplet-based microfluidic platforms for high-throughput screening (HTS). Here, we designed an integrated system based on disulfide-bonded redox-responsive hydrogel beads (redox-HBs), which were prepared through enzymatic hydrogelation, to compartmentalize, screen, select, retrieve, and recover selected Chinese hamster ovary (CHO) cells secreting high levels of antibodies. Moreover, redox-HBs were functionalized with protein G as an antibody-binding module to capture antibodies secreted from encapsulated cells. As proof-of-concept, cells co-producing immunoglobulin G (IgG) as the antibody and green fluorescent protein (GFP) as the reporter molecule, denoted as CHO(IgG/GFP), were encapsulated into functionalized redox-HBs. Additionally, antibody-secreting cells were labeled with protein L-conjugated horseradish peroxidase using a tyramide amplification system, enabling fluorescence staining of the antibody captured inside the beads. Redox-HBs were then applied to fluorescence-activated droplet sorting, and selected redox-HBs were degraded by reducing the disulfide bonds to recover the target cells. The results indicated the potential of the developed HTS platform for selecting a single cell viable for biopharmaceutical production.

Design of Protease-Responsive Antifungal Liposomal Formulation Decorated with a Lipid-Modified Chitin-Binding Domain.

Candida albicans is a prevalent fungal pathogen that displays antibiotic resistance. The polyene antifungal amphotericin B (AmB) has been the gold standard because of its broad antifungal spectra, and its liposomal formulation, AmBisome, has been used widely and clinically in treating fungal infections. Herein, we explored enhancing the antifungal activity of AmBisome by integrating a small chitin-binding domain (LysM) of chitinase A derived from Pteris ryukyuensis. LysM conjugated with a lipid (LysM-lipid) was initially prepared through microbial transglutaminase (MTG)-mediated peptide tag-specific conjugation of LysM with a lipid-peptide substrate. The AmBisome formulation modified with LysM-lipid conjugates had a size distribution that was comparable to the native liposomes but an increased zeta potential, indicating that LysM-lipid conjugates were anchored to AmBisome. LysM-lipid-modified AmBisome exhibited long-term stability at 4 °C while retaining the capacity to bind chitin. Nevertheless, the antifungal efficacy of LysM-lipid-modified AmBisome against C. albicans was modest. We then redesigned a new LysM-lipid conjugate by introducing a peptide linker containing a thrombin digestion (TD) site at the C-terminus of LysM (LysM-TD linker-lipid), thereby facilitating the liberation of the LysM domain from AmBisome upon the addition of thrombin. This new AmBisome formulation anchored with LysM-TD linker-lipid exhibited superior performance in suppressing C. albicans growth in the presence of thrombin compared with the LysM-lipid formulation. These results provide a platform to design stimuli-responsive AmBisome formulations that respond to external environments and thus advance the treatment of pathogenic fungi infections.

Ionic liquid-mediated ethosome for transdermal delivery of insulin.

Herein, we report ethosome (ET) formulations composed of a safe amount of 1,2-dimyristoyl-sn-glycero-3-phosphatidylcholine (DMPC)-based ionic liquid with various concentrations of ethanol as a carrier for the transdermal delivery of a high molecular weight drug, insulin. The Insulin-loaded ET vesicles exhibited long-term stability compared to conventional DMPC ETs, showing significantly higher drug encapsulation efficiency and increased skin permeation ability.

Engineering the Propeptide of Microbial Transglutaminase Zymogen: Enabling Substrate-Dependent Activation for Bioconjugation Applications.

Microbial transglutaminase (MTG) from Streptomyces mobaraensis is a powerful biocatalytic glue for site-specific cross-linking of a range of biomolecules and synthetic molecules that have an MTG-reactive moiety. The preparation of active recombinant MTG requires post-translational proteolytic digestion of a propeptide that functions as an intramolecular chaperone to assist the correct folding of the MTG zymogen (MTGz) in the biosynthesis. Herein, we report engineered active zymogen of MTG (EzMTG) that is expressed in soluble form in the host Escherichia coli cytosol and exhibits cross-linking activity without limited proteolysis of the propeptide. We found that the saturation mutagenesis of residues K10 or Y12 in the propeptide domain generated several active MTGz mutants. In particular, the K10D/Y12G mutant exhibited catalytic activity comparable to that of mature MTG. However, the expression level was low, possibly because of decreased chaperone activity and/or the promiscuous substrate specificity of MTG, which is potentially harmful to the host cells. The K10R/Y12A mutant exhibited specific substrate-dependent reactivity toward peptidyl substrates. Quantitative analysis of the binding affinity of the mutated propeptides to the active site of MTG suggested an inverse relationship between the binding affinity and the catalytic activity of EzMTG. Our proof-of-concept study provides insights into the design of a new biocatalyst using the MTGz as a scaffold and a potential route to high-throughput screening of EzMTG mutants for bioconjugation applications.

A Functional Hydrogel Bead-Based High-Throughput Screening System for Mammalian Cells with Enhanced Secretion of Therapeutic Antibodies.

Droplet-based high-throughput screening systems are an emerging technology that provides a quick test to screen millions of cells with distinctive characteristics. Biopharmaceuticals, specifically therapeutic proteins, are produced by culturing cells that secrete heterologous recombinant proteins with different populations and expression levels; therefore, a technology to discriminate cells that produce more target proteins is needed. Here, we present a droplet-based microfluidic strategy for encapsulating, screening, and selecting target cells with redox-responsive hydrogel beads (HBs). As a proof-of-concept study, we demonstrate the enrichment of hybridoma cells with enhanced capability of antibody secretion using horseradish peroxidase (HRP)-catalyzed hydrogelation of tetra-thiolate poly(ethylene glycol); hybridoma cells were encapsulated in disulfide-bonded HBs. Recombinant protein G or protein M with a C-terminal cysteine residue was installed in the HBs via disulfide bonding to capture antibodies secreted from the cells. HBs were fluorescently stained by adding the protein L-HRP conjugate using a tyramide signal amplification system. HBs were then separated by fluorescence-activated droplet sorting and degraded by reducing the disulfide bonds to recover the target cells. Finally, we succeeded in the selection of hybridoma cells with enhanced antibody secretion, indicating the potential of this system in the therapeutic protein production.

Non-Invasive Transdermal Delivery of Antisense Oligonucleotides with Biocompatible Ionic Liquids.

Nucleic acid drugs, including antisense oligonucleotides (ASOs), have received considerable attention as novel therapeutics for the treatment of intractable diseases. Despite their potential benefits, ASOs are currently administered via injection, which can negatively impact patient quality of life because of the prevalence of severe injection site reactions. Non-invasive transdermal administration of ASOs is desirable but highly challenging owing to the strong barrier imposed by the stratum corneum, which only permits the penetration of small molecules under 500 Da. For ASOs to exert their antisense effect, they must traverse the negatively charged cell membrane and reach the cytoplasm. In this study, we used the solid-in-oil (S/O) dispersion technology to facilitate the skin permeation of ASOs by coating the drug with a hydrophobic surfactant molecule, specifically lipid-based ionic liquid (IL) surfactants with high biocompatibility and transdermal penetration-enhancing properties. To induce the antisense effect, it was important to achieve simultaneous transdermal delivery and intracellular entrapment of ASOs. In vitro investigations indicated that the newly prepared IL-S/O enhanced the transdermal penetration and intracellular delivery of ASOs, thus inhibiting mRNA translation of the target TGF-ß. In addition, in vivo investigations of tumor-bearing mice suggested that the anti-tumor effect of the IL-S/O was similar to that of injection. This study demonstrates the potential of non-invasive transdermal delivery carriers based on biocompatible ILs, which can be applied to a variety of nucleic acid drugs.

Modification with Conventional Surfactants to Improve a Lipid-Based Ionic-Liquid-Associated Transcutaneous Anticancer Vaccine.

Transcutaneous vaccination is one of the successful, affordable, and patient-friendly advanced immunization approaches because of the presence of multiple immune-responsive cell types in the skin. However, in the absence of a preferable facilitator, the skin's outer layer is a strong impediment to delivering biologically active foreign particles. Lipid-based biocompatible ionic-liquid-mediated nanodrug carriers represent an expedient and distinct strategy to permit transdermal drug delivery; with acceptable surfactants, the performance of drug formulations might be further enhanced. For this purpose, we formulated a lipid-based nanovaccine using a conventional (cationic/anionic/nonionic) surfactant loaded with an antigenic protein and immunomodulator in its core to promote drug delivery by penetrating the skin and boosting drug delivery and immunogenic cell activity. In a follow-up investigation, a freeze-dry emulsification process was used to prepare the nanovaccine, and its transdermal delivery, pharmacokinetic parameters, and ability to activate autoimmune cells in the tumor microenvironment were studied in a tumor-budding C57BL/6N mouse model. These analyses were performed using ELISA, nuclei and HE staining, flow cytometry, and other biological techniques. The immunomodulator-containing nanovaccine significantly (p < 0.001) increased transdermal drug delivery and anticancer immune responses (IgG, IgG1, IgG2, CD8+, CD207+, and CD103+ expression) without causing cellular or biological toxicity. Using a nanovaccination approach, it is possible to create a more targeted and efficient delivery system for cancer antigens, thereby stimulating a stronger immune response compared with conventional aqueous formulations. This might lead to more effective therapeutic and preventative outcomes for patients with cancer.

Amino Acid Leaching of Critical Metals from Spent Lithium-Ion Batteries Followed by Selective Recovery of Cobalt Using Aqueous Biphasic System.

To reduce the exploitation of mine resources and decrease the harm to the environment caused by urban electronic wastes, the recovery of critical metals in secondary resources is crucial. In this study, we have successfully developed an eco-friendly process to integrate the leaching and separation of cobalt (Co) from a spent lithium-ion battery (LIB) cathode using an amino acid-based aqueous biphasic system (ABS). We, for the first time, demonstrated a simple method for leaching a LIB cathode using only amino acids. In addition, we have investigated the leaching mechanism using the typical cathode active material lithium cobalt oxide (LiCoO2). Then, the Co was selectively extracted by a biphasic system (amino acid-PPG400-H2O). This novel process has an excellent prospect in the field of spent-battery recycling because of its eco-friendly and process-simplified advantages.

Supramolecular localization in liquid-liquid phase separation and protein recruitment in confined droplets.

This study investigated the localization of artificial peptide supramolecular fibers in liquid-liquid phase separation (LLPS). Hierarchical organization led to the localization of supramolecules in LLPS droplets. Moreover, proteins were recruited into confined droplets by the physical adsorption of proteins on the supramolecules, enabling an enhanced cascade reaction.

Molecular crowding elicits the acceleration of enzymatic crosslinking of macromolecular substrates.

Cytoplasm contains high concentrations of biomacromolecules. Protein behavior under such crowded conditions is reportedly different from that in an aqueous buffer solution, mainly owing to the effect of volume exclusion caused by the presence of macromolecules. Using a crosslinking reaction catalyzed by microbial transglutaminase (MTG) as a model, we herein systematically determined how the substrate size affects enzymatic activity in both dilute and crowded solutions of dextran. We first observed a threefold reduction in MTG-mediated crosslinking of a pair of small peptide substrates in 15 wt% dextran solution. In contrast, when proteinaceous substrates were involved, the crosslinking rates in 15 wt% dextran solutions accelerated markedly to levels comparable with the level in the absence of dextran. Our results provide new insights into the action of enzymes with regard to macromolecular substrates under crowded conditions, of which the potential utility was demonstrated by the formation of highly crosslinked protein polymers.

Transdermal Transmission Blocking Vaccine for Malaria using a Solid-in-Oil Dispersion.

Malaria is a mosquito-borne infectious disease that is widespread in developing countries. Malaria vaccines are important in efforts to eradicate malaria; however, vaccines are usually administered by injection, which requires medical personnel and has a risk of causing infection. Transdermal vaccines can be administered without damaging the skin and thus are ideal for the prevention of malaria. However, the stratum corneum forms a "brick and mortar" like structure in which stratum corneum cells are embedded in a hydrophobic matrix composed of lipids, which strongly inhibits the permeation of hydrophilic substances. In the present study, we designed a transdermal vaccine against vivax malaria using a solid-in-oil (S/O) dispersion. The S/O dispersion of a transmission blocking vaccine candidate, Pvs25 from Plasmodium vivax, showed higher skin penetration than that of the aqueous solution. Mice immunized with the S/O dispersion generated antibodies at similar titers as the mice immunized by injection, over the mid- to long-term. These results provide information for the development of transdermally administered malaria vaccines toward the eradication of malaria.

Transformation of Hydrophilic Drug into Oil-Miscible Ionic Liquids for Transdermal Drug Delivery.

The transdermal delivery of hydrophilic drugs remains challenging owing to their poor ability to permeate the skin; formulation with oil media is difficult without adding chemical permeation enhancers or co-solvents. Herein, we synthesized 12 oil-miscible ionic liquid (IL) drugs comprising lidocaine-, imipramine-, and levamisole (Lev)-hydrochloride with fatty acid permeation enhancers, i.e., laurate, oleate, linoleate, and stearate as counterions. A set of in vitro and in vivo studies was performed to investigate the potency and deliverability of the transdermal drug formulations. All of the synthesized compounds were freely miscible with pharmaceutically acceptable solvents/agents (i.e., ethanol, N-methyl pyrrolidone, Tween 20, and isopropyl myristate (IPM)). In vitro permeation studies revealed that the oleate-based Lev formulation had 2.6-fold higher skin permeation capability than the Lev salts and also superior ability compared with the laurate-, linoleate-, and stearate-containing samples. Upon in vivo transdermal administration to mice, the peak plasma concentration, elimination half-life, and area under the plasma concentration curve values of Lev-IL were 4.6-, 2.9-, and 5.4-fold higher, respectively, than those of the Lev salt. Furthermore, in vitro skin irritation and in vivo histological studies have demonstrated that Lev-IL has excellent biocompatibility compared with a conventional ionic liquid-based carrier. The results indicate that oil-miscible IL-based drugs provide a simple and scalable strategy for the design of effective transdermal drug delivery systems.

Protein-structure-dependent spectral shifts of near-infrared photoluminescence from locally functionalized single-walled carbon nanotubes based on avidin-biotin interactions.

Single-walled carbon nanotubes (SWCNTs) emit photoluminescence (PL) in the near-infrared (NIR) region (>900 nm). To enhance their PL properties, defect doping via local chemical functionalization has been developed. The locally functionalized SWCNTs (lf-SWCNTs) emit red-shifted and bright E11* PL originating from the excitons localized at the defect-doped sites. Here, we observe the E11* PL energy shifts induced by protein adsorption via the avidin-biotin interactions at the doped sites of lf-SWCNTs. We establish that the difference in the structures of the avidin derivatives notably influences the energy shifts. First, lf-SWCNT-tethering biotin groups (lf-SWCNTs-b) are synthesized based on diazonium chemistry, followed by post-modification. The responsiveness of the lf-SWCNTs-b to different microenvironments is investigated, and a correlation between the E11* PL energy shift and the induction-polarity parameters of surrounding solvents is established. The adsorption of neutravidin onto the lf-SWCNTs-b induces an increase in the induction-polarity parameters around the biotin-doped sites, resulting in the red-shift of the E11* PL peak. The E11* PL shift behaviors of the lf-SWCNTs-b change noticeably when avidin and streptavidin are introduced compared to the case with neutravidin. This is due to the different microenvironments formed at the biotin-doped sites, attributed to the difference in the structural features of the introduced avidin derivatives. Moreover, we successfully enhance the detection signals of lf-SWCNTs-b (>three fold) for streptavidin detection using a fabricated film device. Therefore, lf-SWCNTs exhibit significant promise for application in advanced protein detection/recognition devices based on NIR PL.

Artificial Palmitoylation of Proteins Controls the Lipid Domain-Selective Anchoring on Biomembranes and the Raft-Dependent Cellular Internalization.

Protein palmitoylation, a post-translational modification, is universally observed in eukaryotic cells. The localization of palmitoylated proteins to highly dynamic, sphingolipid- and cholesterol-rich microdomains (called lipid rafts) on the plasma membrane has been shown to play an important role in signal transduction in cells. However, this complex biological system is not yet completely understood. Here, we used a combined approach where an artificial lipidated protein was applied to biomimetic model membranes and plasma membranes in cells to illuminate chemical and physiological properties of the rafts. Using cell-sized giant unilamellar vesicles, we demonstrated the selective partitioning of enhanced green fluorescent protein modified with a C-terminal palmitoyl moiety (EGFP-Pal) into the liquid-ordered phase consisting of saturated phospholipids and cholesterol. Using Jurkat T cells treated with an immunostimulant (concanavalin A), we observed the vesicular transport of EGFP-Pal. Further cellular studies with the treatment of methyl ß-cyclodextrin revealed the cholesterol-dependent internalization of EGFP-Pal, which can be explained by a raft-dependent, caveolae-mediated endocytic pathway. The present synthetic approach using artificial and natural membrane systems can be further extended to explore the potential utility of artificially lipidated proteins at biological and artificial interfaces.

Transdermal Delivery of Antigenic Protein Using Ionic Liquid-Based Nanocarriers for Tumor Immunotherapy.

Transdermal drug delivery systems (TDDSs) may be useful for preventing various diseases including cancer. However, the stratum corneum (SC) inhibits the permeation of foreign particles into the skin. To obtain an effective TDDS, we developed a protein-containing nanocarrier (PCNC) comprising an antigenic protein (ovalbumin/OVA) stabilized by a combination of surfactants, i.e., a lipid-based surface-active ionic liquid and Tween-80. The PCNC was lyophilized to remove water and cyclohexane and then dispersed in isopropyl myristate. It is biocompatible both in vitro and in vivo, and is suitable for use in a therapeutic TDDS. The skin permeability of the PCNC was significantly (p < 0.0001) enhanced, and the transdermal distribution and transdermal flux of the OVA delivery system were 25 and 28 times greater, respectively, than those of its aqueous formulation. The PCNC disrupted the order of lipid orientation in the skin's SC and increased intercellular protein delivery. It demonstrated effective antitumor activity, drastically (p < 0.001) suppressed tumor growth, increased mouse survival rates, and significantly (p < 0.001) stimulated the OVA-specific tumor immune response. The PCNC also increased the number of cytotoxic T cells expressing CD8 antibodies on their surfaces (CD8 + T-cells) in the tumor microenvironment. These findings suggest that PCNCs may be promising biocompatible carriers for transdermal antigenic protein delivery in tumor immunotherapy.

A solid-in-oil-in-water emulsion: An adjuvant-based immune-carrier enhances vaccine effect.

The biomaterial-based immunoengineering has become one of the most attractive research fields in the last decade. In the present study, a solid-in-oil-in-water (S/O/W) emulsion encapsulating antigen in the oil phase of an oil-in-water (O/W) emulsion was prepared as a novel vaccine carrier consisting of similar materials to the emulsion adjuvant of which the safety, immunogenicity and vaccination efficacy have been already confirmed in human. Direct observation by high-resolution confocal laser scanning microscopy and small angle X-ray scattering analysis showed that the antigens were dispersed inside of the oil phase of the S/O/W emulsion as solid-state particles. The S/O/W emulsion robustly produced antigen-specific antibodies and enhanced the antitumor effects in a therapeutic cancer vaccination compared with free antigens or the O/W emulsion in vivo. This result is in good agreement with the activation effect of antigen-specific cytotoxic T lymphocytes and antigen presentation by the S/O/W emulsion, indicating that the S/O/W emulsion consisting of already approved materials is a promising vaccine carrier to produce both humoral and cellular immunity.

Hydrophobic immiscibility controls self-sorting or co-assembly of peptide amphiphiles.

Pairs of peptide amphiphiles with immiscible hydrophobic tails were synthesized and their assembly formation was investigated. These pairs formed self-sorting supramolecular fibres using a standard heating-cooling protocol, while one pair with longer hydrophobic tails formed a co-assembly when an additional heating process was applied.

Design of Swollen Lipidic Cubic Phase to Increase Transcutaneous Penetration of Biomacromolecules.

Lipidic cubic phase (LCP) is a self-assembled system composed of lipids with interpenetrated aqueous channels, and its potential in drug delivery systems has been investigated. Although LCP was shown to improve transcutaneous penetration of hydrophilic molecules of up to 1203 Da so far, the transcutaneous delivery of larger molecules such as proteins has not been achieved. This is likely because proteins are usually larger than the aqueous channels of LCP (∼37.2 Å in diameter), which limits the molecular diffusion in LCP. In this report, we overcome this issue by adding N-octyl-ß-d-glucopyranoside to glyceryl monooleate-water-based LCP to give swollen LCP (SLCP), which has larger aqueous channel diameters (∼65.6 Å). First, we systemically evaluated the effect of swelling on drug diffusion in LCP/SLCP. The release kinetics of various peptides and proteins whose sizes ranged from 9.14 to 55.28 Å in diameter were evaluated, and the diffusion coefficients (D) were calculated by the Fickian diffusion model. As expected, all peptides and proteins diffused faster in SLCP than in LCP. A more detailed analysis revealed a negative linear relationship between log D and the ratio of the radius of gyration of the proteins to the aqueous channel radius, indicating that swelling of a cubic nanostructure is an effective strategy to enhance D. Next, the skin penetration of proteins encapsulated in LCP and SLCP was evaluated. The skin penetration of ovalbumin (42.9 kDa), for example, was enhanced by SLCP but not by LCP, and a positive correlation between D and the amount of skin penetration was found. Collectively, this study provides an effective measure for designing LCP systems that enhance transcutaneous penetration of biomacromolecules.

Methotrexate-based ionic liquid as a potent anticancer drug for oral delivery: In vivo pharmacokinetics, biodistribution, and antitumor efficacy.

Oral delivery of the sparingly soluble drug methotrexate (MTX) is challenging owing to its poor bioavailability and low solubility. To address this challenge, the present study reports the conversion of MTX into a series of five ionic liquids (ILs) comprising a cationic component-i.e., cholinium (Cho), tetramethylammonium (TMA), tetrabutylphosphonium (TBP), or an amino acid ester-and an anionic component-i.e., MTX. The biocompatibility, pharmacokinetics, tissue distribution, and antitumor efficacy of each MTX-based IL were investigated to determine its usefulness as a pharmaceutical. Oral administration to mice revealed that proline ethyl ester MTX (IL[ProEt][MTX]) had 4.6-fold higher oral bioavailability than MTX sodium, followed by aspartic diethyl ester MTX, IL[TBP][MTX], IL[Cho][MTX], and IL[TMA][MTX]. The peak plasma concentration, elimination half-life, area under the plasma concentration, mean absorption time, and body clearance of IL[ProEt][MTX] were significantly (p < 0.0001) higher by 1.7-, 6.2-, 4.6-, 2.5-, and 3.6-fold, respectively, than those of MTX sodium. MTX accumulation in the lungs, spleen, kidney, and gastrointestinal tract was also reduced by 5.6-, 1.8-, 1.5-, and 1.4-fold, respectively, indicating the IL formulations had lower systemic toxicity than free MTX. Mechanistic studies revealed that the IL[ProEt][MTX] solution formed spherical structures with an average size of 190 nm. This was probably responsible for its improved oral absorption performance in vivo. In vivo antitumor studies also demonstrated that IL[ProEt][MTX] suppressed tumor growth more than MTX sodium. These results suggest that MTX-based ILs provide a simple scalable approach to improving the oral bioavailability of poorly soluble MTX.