Spotlighting rising researcher Mikaela Gray: one ingredient intracellular delivery of RNA by hydrophobic ion pairing

About the impact of superassociation of hydrophobic ion pairs on membrane permeability

Ethyl lauroyl arginate-based hydrophobic ion pair complex in lipid nanocapsules: A novel oral delivery approach of rosmarinic acid for enhanced permeability and bioavailability

Peptide drug delivery: Permeation behaviour and intracellular fate of hydrophobic ion pairs in self-emulsifying drug delivery systems.

Hypothesis: Hydrophobic ion pairs (HIPs) between two fluorescent components and incorporation into nanoemulsions (NE) allows tracking in cellular uptake studies.Experiments: HIPs were formed between propidium iodide and 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-(7-nitro-2–1,3-benzoxadiazol-4-yl) (NBD-PE), azure A chloride and NBD-PE or coumarin 343 and 4-(4-dihexadecylaminostyryl)-N-methylpyridinium iodide) (DiA). Fluorescence spectra of the resulting complexes were recorded. HIPs were loaded into zwitterionic NE and their size, stability in different media, haemolytic properties and cytotoxicity were evaluated. Furthermore, cellular uptake at 37 °C and 4 °C was investigated via flow cytometry and confocal microscopy.Findings: HIP-formation increased lipophilicity of the hydrophilic model drugs. NE exhibited a size between 80 and 150 nm and were not toxic in concentrations up to 0.1 % but showed high haemolytic properties. Cellular uptake of propidium, azure A and coumarin 343 were 8-fold, 115-fold and 1.3-fold improved by the formation of HIPs and up to 59-fold, 120-fold and 50-fold by incorporating these HIPs in NE, respectively. Lower uptake was observed at 4 °C. In case of propidium/ NBD-PE and azure A/ NBD-PE HIPs, propidium and azure A were delivered into the cytosol, whereas NBD-PE was unable to enter cells. In case of coumarin 343/ DiA HIPs, both components accumulated in the cell membrane. Therefore, HIPs between two fluorescent compounds are a powerful tool to investigate cellular uptake of hydrophobic complexes via nanocarriers by visualization of their cellular distribution.

Self-emulsifying drug delivery systems: About the fate of hydrophobic ion pairs on a phospholipid bilayer

Proteins can perform ideal therapeutic functions. However, their large size and significant surface hydrophilicity and charge prohibit them from reaching intracellular targets. These chemical features also render them poorly encapsulated by nanoparticles used for intracellular delivery. In this work, a novel combination of protein vesicles and hydrophobic ion pairing (HIP) was used to load protein cargo and achieve cytosolic delivery to overcome the limitations of previous protein vesicle properties. Protein vesicles are thermally self-assembling nanoparticles made from elastin-like polypeptide (ELP) fused to an arginine-rich leucine zipper and a globular protein fused to a glutamate-rich leucine zipper. To impart stimuli-responsive disassembly, physiological stability, and small size, the ELP sequence was modified to include histidine and tyrosine residues. HIP was used to load and release protein cargo requiring endosomal escape for cytosolic function. HIP vesicles enabled delivery of cytochrome c, a cytosolically active protein, and a significant reduction in viability in both a traditional two-dimensional (2D) human cancer cell line culture and a biomimetic three-dimensional (3D) organoid model of acute myeloid leukemia. By examining the uptake of positively and negatively charged fluorescent protein cargos loaded by HIP, this work revealed the necessity of HIP for cytosolic cargo delivery and how HIP loading influences protein vesicle self-assembly and disassembly using microscopy, small-angle X-ray scattering, and nanoparticle tracking analysis. HIP protein vesicles have the potential to broaden the use of intracellular proteins as therapeutics for various diseases and extend protein vesicles to deliver other biomacromolecules, as the strategy developed here resulted in the first cytosolic protein cargo delivery using protein vesicles.

Hydrophobic ion pairing has emerged as a method to modulate the solubility of charged hydrophilic molecules ranging in class from small molecules to large enzymes. Charged hydrophilic molecules are ionically paired with oppositely-charged molecules that include hydrophobic moieties; the resulting uncharged complex is water-insoluble and will precipitate in aqueous media. Here we review one of the most prominent applications of hydrophobic ion pairing: efficient encapsulation of charged hydrophilic molecules into nano-scale delivery vehicles – nanoparticles or nanocarriers. Hydrophobic complexes are formed and then encapsulated using techniques developed for poorly-water-soluble therapeutics. With this approach, researchers have reported encapsulation efficiencies up to 100% and drug loadings up to 30%. This review covers the fundamentals of hydrophobic ion pairing, including nomenclature, drug eligibility for the technique, commonly-used counterions, and drug release of encapsulated ion paired complexes. We then focus on nanoformulation techniques used in concert with hydrophobic ion pairing and note strengths and weaknesses specific to each. The penultimate section bridges hydrophobic ion pairing with the related fields of polyelectrolyte coacervation and polyelectrolyte-surfactant complexation. We then discuss the state of the art and anticipated future challenges. The review ends with comprehensive tables of reported hydrophobic ion pairing and encapsulation from the literature.

Self-emulsifying drug delivery systems: Impact of stability of hydrophobic ion pairs on drug release

It was the aim of this study to evaluate the potential of reverse micelles (RM) and hydrophobic ion pairs (HIP) for incorporation of semaglutide into self-emulsifying oral drug delivery systems. Reverse micelles loaded with semaglutide were formed with a cationic (ethyl lauroyl arginate, ELA) and an anionic surfactant (docusate, DOC), whereas HIP were formed between semaglutide and ELA. Maximum solubility of the peptide and the rate of dissolution was evaluated in various lipophilic phases (glycerol monocaprylocaprate:caprylic acid 1:4 (m/m), glycerol monolinoleate:caprylic acid 1:4 (m/m) and glycerol monocaprylocaprate:glycerol monolinoleate 1:4 (m/m)). Self-emulsifying drug delivery systems (SEDDS) loaded with RM and HIP were characterized regarding size distribution, zeta potential, cytocompatibility and Caco-2 permeability. Droplet sizes between 50 and 300 nm with polydispersity index (PDI) around 0.3 and zeta potentials between − 45 mV (RMDOC) and 36 mV (RMELA) were obtained. RM provided an almost 2-fold higher lipophilicity of semaglutide than HIP resulting in a 4.2-fold higher payload of SEDDS compared to HIP. SEDDS containing RM or HIP showed high cytocompatibilities with a cell survival above 75% for concentrations up to 0.1% on Caco-2 cells and acceptable hemolytic activity. Permeation studies across Caco-2 monolayer revealed an at least 2-fold increase in permeability of semaglutide for the developed formulations.

The combination of Flash NanoPrecipitation and hydrophobic ion pairing (HIP) is a valuable approach for generating nanocarrier formulations of ionic water-soluble drugs with controllable release properties dictated by liquid crystalline structuring of the ion pairs. However, there are few examples of this in practice in the literature. This work aims to decipher the influence of the nature of the hydrophobic counterion used in HIP and its consequent impact on liquid crystalline structuring and drug release. The hypothesis of this study was that hydrophobic counterions with different head and tail groups used for FNP with HIP would give rise to different liquid crystalline structures, which in turn would result in different drug release behavior. A cationic, water-soluble antibiotic, polymixin B, was complexed with eight different hydrophobic counterions with varying head and tail groups and encapsulated into nanocarriers 100-400 nm in size prepared using FNP. Sixteen formulations were assessed for internal structure by synchrotron small-angle X-ray scattering, and drug release was measured in vitro in physiological conditions. The liquid crystalline phases formed depended on the counterion head group and tail geometry, drug:counterion charge ratio, and the ionic strength and pH of the release medium. Drug release occurred more rapidly when no liquid crystalline phases were present and more slowly when higher-ordered phases existed. Specific findings include that phosphonic acid counterions lead to the formation of lamellar structures that persisted at pH 2.0 but were not present at pH 7.3. In contrast, sulfonic acids lead to lamellar or hexagonal phases that persisted at both pH 7.3 and 2.0, while hydrophobic counterions without alkyl tails did not form internal structures. It was also clear that the lipophilicity of the counterion does not dictate drug release. These findings confirm that the liquid crystalline phase behavior of the drug:counterion complex dictates drug release and significantly improves our understanding of the types of controlled release formulations that are possible using FNP with HIP.

Biopharmaceutical classification systems (BCS) class III drugs belongs to a group of drugs with high solubility in gastrointestinal (GI) fluids and low membrane permeability result in significantly low bioavailability. Self-emulsifying drug delivery systems (SEDDS) considered a suitable candidate to enhance the bioavailability of poorly soluble drugs by improving their membrane permeability, however, incorporating hydrophilic drugs in to these carriers remained a great challenge. The aim of this study was to develop hydrophobic ion pairs (HIPs) of a model BCS class-III drug tobramycin (TOB) in order to incorporate into SEDDS and improve its bioavailability. HIPs of TOB were formulated using anionic surfactants sodium docusate (DOC) and sodium dodecanoate (DOD). The efficiency of HIPs was estimated by measuring the concentration of formed complexes in water, zeta potential determination and log P value evaluation. Solubility studies of HIPs of TOB with DOC were accomplished to screen the suitable excipients for SEDDS development. Consequently, HIPs of TOB with DOC were loaded into SEDDS and assessed the log DSEDDS/release medium and dissociation of these complexes at different intestinal pH over time. Moreover, cytotoxic potential of HIPs of TOB and HIPs loaded SEDDS formulations was evaluated. HIPs of TOB with DOC exhibited the maximum precipitation efficiency at a stoichiometric ratio of 1:5. Log P of HIPs of TOB improved up to 1500-fold compared to free TOB. Zeta potential of TOB was shifted from positive to negative during hydrophobic ion pairing (HIP). HIPs of TOB with DOC was loaded at a concentration of 1% (w/v) into SEDDS formulations. Log DSEDDS/release medium of loaded complexes in to oily droplets was above 2 and dissociated up to 20% at various pH within 4 h. Finding of this study suggested that improvement of the lipophilic character of BCS class-III drugs followed by incorporation into oily droplets can be deliberated as a promising tool to enhance the permeation across biological membranes.

Impact of different hydrophobic ion pairs of octreotide on its oral bioavailability in pigs

A novel in situ hydrophobic ion pairing (HIP) formulation strategy for clinical product selection of a nanoparticle drug delivery system

Background: Hydrophobic ion pairing is a technique for reducing the hydrophilicity of charged molecules (drugs) by pairing them with oppositely charged hydrophobic counterions. This method is used to control the solubility of charged molecules in a solvent and is of particular importance in drug delivery. Methods: Dissipative particle dynamics simulations were performed to provide a microscopic understanding of hydrophobic ion pairing in polymyxin B (PMB) and oleate (OA) ions. Solvents and ions were explicitly included in the simulations. Results: We investigated the effects of relative concentrations of PMB and OA (the charge ratio), solvent philicity, and the concentrations of PMB and OA at a fixed composition on the structural stability and the hydrophobicity of the ion paired cluster, as well as the kinetics of assembly. The maximum hydrophobicity belongs to PMB:OA charge ratio 1:1. The clustering efficiency in mixed ethanol-water solutions decreases with the increasing ethanol content of water. The dynamics of PMB/OA exchange between hydrophobic cluster and the surrounding solution reveal two distinct relaxation processes, whose relaxation times differ by two orders of magnitude. Conclusions: The hydrophobicity of the cluster is controlled by the charge ratio. The core of the ion paired cluster acts as the primary barrier and its surface layer acts as the secondary barrier against alcohol permeation into it. The exchange of surface PMB/OA ions with the surrounding is a much faster dynamic process than the establishment of equilibrium between the PMB/OA ions in the cluster and the solution. The time scale for the slower process provides useful information on the rate of drug release from the hydrophobic ion paired complex.

The present work aimed to form hydrophobic ion pairs(HIPs) ofa small molecule remaining inside the oily droplets of SEDDS to ahigh extent. HIPs of ethacridine and various surfactants classifiedby functional groups of phosphates, sulfates, and sulfonates wereformed and precipitation efficiency, log Dn-octanol/water, and solubility in different excipientswere investigated. Most lipophilic HIPs were incorporated into SEDDSand evaluated regarding drug release. Docusate HIPs showed the highestincrease in lipophilicity with a precipitation efficiency of 100%,a log Dn-octanol/water of2.66 and a solubility of 132 mg/mL in n-octanol,123 mg/mL in oleyl alcohol, and 40 mg/mL in medium chain triglycerides.Docusate HIPs were incorporated into three SEDDS of increasing lipophilicity(F1 < F2 < F3) based on medium chain triglycerides, oleyl alcohol,Kolliphor EL, and Tween 80 (F1: 1 + 5 + 2 + 2; F2: 3 + 3 + 2 + 2;F3: 5 + 1 + 4 + 0). Highest achievable payloads ranged from 74.49mg/mL (F3) to 97.13 mg/mL (F1) and log DSEDDS/RM increased by at least 7.5 units (4.99, F1). Drug release studiesvia the diffusion membrane method confirmed minor release of docusateHIPs from all SEDDS (<2.7% within 4 h). In conclusion, highly lipophilicHIPs remain inside the oily phase of SEDDS and likely reach the absorptionmembrane in intact form.