Search Results (34)

Peptide drug development has emerged as a prominent area in pharmaceutical research due to its high specificity and therapeutic potential. However, their biological activity, stability, and bioavailability are significantly influenced by interactions with counter-ions, which electrostatically bind to charged residues on peptide surfaces. This review systematically examines the multifaceted roles of counter-ions in modulating peptide structure and function. Counter-ions are classified into organic/inorganic and anionic/cationic categories, with their selection critically impacting peptide solubility, conformational stability, and activity. Inorganic counter-ions could enhance structural integrity, while organic counter-ions could mitigate toxicity risks. Notably, counter-ions can induce secondary structural transitions, directly affecting biological efficacy. Furthermore, counter-ions play pivotal roles in drug delivery systems, including nanoemulsions, self-emulsifying formulations, and lipid-based nanoparticles, where hydrophobic ion pairing improves encapsulation efficiency and oral bioavailability. In chromatography, ion-pairing reagents optimize peptide separation but may compromise mass spectrometry compatibility. Emerging analytical techniques, such as capillary electrophoresis and liquid chromatography–tandem mass spectrometry (LC-MS/MS), enhance counter-ion detection precision, addressing challenges in pharmaceutical quality control. Despite advancements, gaps remain in understanding ion-specific binding mechanisms and long-term safety profiles. This review underscores the necessity of tailoring counter-ion selection to balance efficacy, stability, and biocompatibility. Future research should prioritize elucidating molecular interaction dynamics and developing safer, high-affinity counter-ions to overcome current limitations in peptide drug development. Full article

Background/Objectives: Oral insulin continues to constitute a challenge due to its low uptake by the gut wall and degradation by gastrointestinal proteolytic enzymes. Such concerns might be surmounted by means of nanoparticle delivery. Methods: In this study, glargine insulin has been loaded into solid lipid nanoparticles prepared via coacervation from Shea and Mango soaps, due to hydrophobic ion pairing. Subsequently, ex vivo tied-up-gut experiments were performed with fluorescently labeled peptide. Additionally, re-dispersible oral solid dosage forms (powders and tablets) were obtained from nanoparticle suspensions via freeze-drying and spray-drying. Results: Solid lipid nanoparticles are capable of enhancing peptide permeation into different gut sections. Furthermore, spray-drying permits the preparation, which can be scaled up, of a re-dispersible powder from the nanoparticle suspension. Conclusions: This engineered process is suitable for the formulation of solid oral dosage forms, such as granulates and tablets, and presents promising potential for oral insulin delivery, paving the way for the assessment of its pharmacological efficacy in further in vivo studies. Full article

Surfactants are used in various washing applications with potential negative environmental and health impacts. The ion-pair 1,3-dioctadecyl-1H-1,2,3-triazol-3-ium-tetraphenylborate (DODTA–TPB) was used to fabricate the potentiometric sensor for the quantification of anionic surfactants. The computational analysis of the DODTA⁺–TPB⁻ adduct reveals a dynamic, thermodynamically favorable interaction driven primarily by hydrophobic C–H∙∙∙π contacts and the flexibility of the C-18 chains, rather than electrostatic or π–π stacking forces. These findings, supported by the MM-PBSA, RDF, and structural analyses, align with broader trends in molecular recognition and provide a foundation for designing advanced ion-pair-based sensors. The sensor showed advanced analytical properties to anionic surfactants with low interfering effects of selected anions. The response of the SDS was investigated in the range from 8.1 × 10⁻⁸ M to 1.0 × 10⁻² M, with a slope of −59.2 mV and a limit of detection (LOD) of 3.1 × 10⁻⁷ M; and DBS was in the range of 8.1 × 10⁻⁸ M to 2.5 × 10⁻³ M with a slope of −57.5 mV and an LOD of 5.9 × 10⁻⁷ M. The sensor was tested on potential interfering ions. Potentiometric titrations of technical-grade anionic surfactants had high recovery rates from 100.2 to 100.4%. The recovery test for spiked samples of surface waters was from 94.2 to 96.5%. The sensor was tested on commercial samples containing anionic surfactants, and the results were compared and showed a good agreement with the two-phase titration method. Full article

Background/Objectives: Leishmaniasis is a prevailing infectious disease transmitted via infected phlebotomine sandflies. The lack of an efficient vaccine with respect to immunogenic antigens and adjuvanted delivery systems impedes its control. Following the induction of immune responses in mice vaccinated with multi-epitope Leishmania peptides (LeishPts) encapsulated in doubly adjuvanted self-nanoemulsifying drug delivery systems (ST-SNEDDSs), this study aims to assess ST-SNEDDS-based nanoemulsions as vehicles for the delivery of antigenic proteins. Methods: Model antigens (e.g., BSA-FITC, OVA) were encapsulated in ST-SNEDDS after being complexed with the cationic phospholipid dimyristoyl phosphatidylglycerol (DMPG) via hydrophobic ion pairing. The nanoemulsions were characterized with respect to droplet diameter, zeta potential, stability, protein loading, protein release from the nanodroplets in different release media and cell uptake. Results: Both model antigens exhibited high encapsulation efficiency (>95%) and their release from the nanodroplets was shown to be strongly affected by the type of release medium (e.g., PBS, FBS 10% v/v) and the ratio of its volume to that of the oily phase, in agreement with predictions of protein release. Protein-loaded nanoemulsion droplets labeled with Cy-5 were found to be efficiently taken up by macrophages (J774A.1) in vitro. However, no colocalization of the labeled nanodroplets and BSA-FITC could be observed. Conclusions: It was revealed that in contrast with LeishPts, whole protein molecules may not be appropriate antigenic cargo for ST-SNEDDS formulations due to the rapid protein release from the nanodroplets in release media simulating in vitro culture and in vivo conditions such as FBS 10% v/v. Full article

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. Full article

Tetrodotoxin (TTX) is a representative natural toxin causing pufferfish food poisoning, which is especially prominent in East and Southeast Asia, including Japan. TTX has been analyzed through post-column derivatization high-performance liquid chromatography (HPLC), ion-pair LC-MS(/MS), and hydrophilic interaction liquid chromatography (HILIC)-MS(/MS) as alternatives to the mouse bioassay method. However, post-column derivatization requires a system for online derivatization reactions, and with the ion-pair LC-MS approach, it is difficult to remove residual ion-pair reagents remaining in the equipment. Moreover, HILIC-MS provides poor separation compared to reversed-phase (RP) HPLC and requires a long time to reach equilibration. Therefore, we decided to develop a TTX analytical method using pre-column derivatization and RP HPLC for the rapid assessment of outbreak samples, including food remnants. In this study, we focused on the vic-diol moiety of TTX and designed a new derivatization reagent coded as NBD-H-DAB. This NBD-H-DAB was synthesized from 4-hydrazino-7-nitro-2,1,3-benzoxadiazole (NBD-H) and 3-fluoro-2-formylphenylboronic acid (FFPBA) with a simple reaction system and rapidly converted to its boronate form, coded NBD-H-PBA, in an aqueous reaction solution. The NBD-H-PBA demonstrated appropriate hydrophobicity to be retained on the RP analytical column and successfully detected with a UV spectrometer. It was easily reacted with the vic-diol moiety of TTX (C6 and C11) to synthesized a boronic ester. The derivatized TTX could be detected using the RP HPLC-UV, and the limit of detection in the fish flesh samples was 0.06 mg/kg. This novel pre-column derivatization of TTX with NBD-H-PBA proves capable for the analysis of TTX. Full article

Anion-driven, nanoscale polar–apolar structural organization is investigated in a solvate ionic liquid (SIL) setting by comparing sulfonate-based anions with long and short perfluorinated alkyl chains. Representative SILs are created from 1,2-bis(2-methoxyethoxy)ethane (“triglyme” or “G3”), lithium nonafluoro-1-butanesulfonate, and lithium trifluoromethanesulfonate. Molecular dynamics simulations, density functional theory computations, and vibrational spectroscopy provide insight into the overall liquid structure, cation–solvent interactions, and cation–anion association. Significant competition between G3 and anions for cation-binding sites characterizes the G3–LiC₄F₉SO₃ mixtures. Only 50% of coordinating G3 molecules form tetradentate complexes with Li⁺ in [(G3)₁Li][C₄F₉SO₃]. Moreover, the SIL is characterized by extensive amounts of ion pairing. Based on these observations, [(G3)₁Li][C₄F₉SO₃] is classified as a “poor” SIL, similar to the analogous [(G3)₁Li][CF₃SO₃] system. Even though the comparable basicity of the CF₃SO₃⁻ and C₄F₉SO₃⁻ anions leads to similar SIL classifications, the hydrophobic fluorobutyl groups support extensive apolar domain formation. These apolar moieties permeate throughout [(G3)₁Li][C₄F₉SO₃] and persist even at relatively low dilution ratios of [(G3)₁₀Li][C₄F₉SO₃]. By way of comparison, the CF₃ group is far too short to sustain polar–apolar segregation. This demonstrates how chemically modifying the anions to include hydrophobic groups can impart unique nanoscale organization to a SIL. Moreover, tuning these nano-segregated fluorinated domains could, in principle, control the presence of dimensionally ordered states in these mixtures without changing the coordination of the lithium ions. Full article

Hydrophobic ion pairing (HIP) complexation was found to be an efficient approach in modulating the release and enhancing the stability and encapsulation of hydrophilic macromolecules such as proteins in hydrophobic nano/microcarriers. The present work strives to develop and optimize the preparation of the HIP complex of the antimicrobial enzyme lysozyme (LYZ) with the ion-pairing agent (IPA) sodium dodecyl sulphate (SDS) relying on the quality-by-design (QbD) approach. The quality target product profile (QTPP) includes the achievement of maximal lipophilicity in a reversible manner to enable the maintenance of biological activity. The related critical quality attributes (CQAs) were defined as complexation efficacy, complex stability, enzyme recovery and activity. Three risk assessment (RA) tools were used to identify and rank the critical process parameters (CPPs) and critical material attributes (CMAs). From this assessment, the pH of the medium, LYZ:SDS molar ratio and drying conditions were determined as high-risk factors that need to be investigated. To the best of our knowledge, for the first time, electrostatic titration was used as a smart approach to determine the optimum molar ratio at different pH values. Based on the predefined CQAs, pH 8 with an LYZ/SDS molar ratio of 1:8 was found to be the optimal condition for complexation efficiency and recovery (%) of a biologically active enzyme. A cost-effective drying process based on a ventilated oven was developed, which resulted in complex qualities comparable to those obtained by the commonly used freeze-drying method. In a nutshell, the optimum conditions for the preparation of the LYZ/SDS HIP complex were efficiently facilitated by the rational application of QbD principles and the utilization of efficient electrostatic titration and ventilated oven-drying methods. Full article

Poor prognosis in high-grade gliomas is mainly due to fatal relapse after surgical resection in the absence of efficient chemotherapy, which is severely hampered by the blood–brain barrier. However, the leaky blood–brain–tumour barrier forms upon tumour growth and vascularization, allowing targeted nanocarrier-mediated drug delivery. The homotypic targeting ability of cell-membrane fragments obtained from cancer cells means that these fragments can be exploited to this aim. In this experimental work, injectable nanoemulsions, which have a long history of safe clinic usage, have been wrapped in glioma-cell membrane fragments via co-extrusion to give targeted, homogeneously sized, sterile formulations. These systems were then loaded with three different chemotherapeutics, in the form of hydrophobic ion pairs that can be released into the target site thanks to interactions with physiological components. The numerous assays performed in two-dimensional (2D) and three-dimensional (3D) cell models demonstrate that the proposed approach is a versatile drug-delivery platform with chemo-tactic properties towards glioma cells, with adhesive interactions between the target cell and the cell membrane fragments most likely being responsible for the effect. This approach’s promising translational perspectives towards personalized nanomedicine mean that further in vivo studies are foreseen for the future. Full article

The possibilities of comparing computational results of noncovalent interactions with experimental data are discussed, first with respect to intramolecular interactions. For these a variety of experimental data such as heats of formation, crystal sublimation heats, comparison with energy minimized structures, and spectroscopic data are available, but until now largely have not found widespread application. Early force field and QM/MP2 calculations have already shown that the sublimation heats of hydrocarbons can be predicted with an accuracy of ±1%. Intermolecular interactions in solution or the gas phase are always accompanied by difficult to compute entropic contributions, like all associations between molecules. Experimentally observed T∆S values contribute 10% to 80% of the total ∆G, depending on interaction mechanisms within the complexes, such as, e.g., hydrogen bonding and ion pairing. Free energies ∆G derived from equilibrium measurements in solution allow us to define binding increments ∆∆G, which are additive and transferable to a variety of supramolecular complexes. Data from more than 90 equilibrium measurements of porphyrin receptors in water indicate that small alkanes do not bind to the hydrophobic flat surfaces within a measuring limit of ∆G = ±0.5 kJ/mol, and that 20 functions bearing heteroatoms show associations by dispersive interactions with up to ∆G = 8 kJ/mol, roughly as a function of their polarizability. Aromatic systems display size-dependent affinities ∆G as a linear function of the number of π-electrons. Full article

Despite several novel and innovative approaches, clinical translation of oral insulin delivery into commercially viable treatment is still challenging due to its poor absorption and rapid degradation in GIT. Thus, an insulin-SDS hydrophobic ion pair loaded self-microemulsifying drug delivery system (SMEDDS) was formulated to exploit the hypoglycemic effects of orally delivered insulin. Insulin was initially hydrophobically ion paired with sodium dodecyl sulphate (SDS) to enhance its lipophilicity. The successful complexation of Insulin-SDS was confirmed by FTIR and surface morphology was evaluated using SEM. Stability of insulin after its release from HIP complex was evaluated using SDS PAGE. Subsequently, Ins-SDS loaded SMEDDS was optimized using two factorial designs. In vitro stability of insulin entrapped in optimized SMEDDS against proteolytic degradation was also assessed. Further, antidiabetic activity of optimized Ins-SDS loaded SMEDDS was evaluated in diabetic rats. Insulin complexed with SDS at 6:1 (SDS/insulin) molar ratio with almost five-fold increased lipophilicity. The SMEDDS was optimized at 10% Labraphil M2125 CS, 70% Cremophore EL, and 20% Transcutol HP with better proteolytic stability and oral antidiabetic activity. An Ins-SDS loaded SMEDDS was successfully optimized. Compared with insulin and Ins-SDS complex, the optimized SMEDDS displayed considerable resistance to GI enzymes. Thus, the SMEDDS showed potential for effective delivery of macromolecular drugs with improved oral bioavailability. Full article

The histidine-containing phosphocarrier (HPr) is a monomeric protein conserved in Gram-positive bacteria, which may be of mesophilic or thermophilic nature. In particular, the HPr protein from the thermophilic organism B. stearothermophilus is a good model system for thermostability studies, since experimental data, such as crystal structure and thermal stability curves, are available. However, its unfolding mechanism at higher temperatures is yet unclear at a molecular level. Therefore, in this work, we researched the thermal stability of this protein using molecular dynamics simulations, subjecting it to five different temperatures during a time span of 1 μs. The analyses of the structural parameters and molecular interactions were compared with those of the mesophilic homologue HPr protein from B. subtilis. Each simulation was run in triplicate using identical conditions for both proteins. The results showed that the two proteins lose stability as the temperature increases, but the mesophilic structure is more affected. We found that the salt bridge network formed by the triad of Glu3-Lys62-Glu36 residues and the salt bridge made up of Asp79-Lys83 ion pair are key factors to keep stable the thermophilic protein, maintaining the hydrophobic core protected and the structure packed. In addition, these molecular interactions neutralize the negative surface charge, acting as “natural molecular staples”. Full article

Flash nanoprecipitation (FNP) is a turbulent mixing process capable of reproducibly producing polymer nanoparticles loaded with active pharmaceutical ingredients (APIs). The nanoparticles produced with this method consist of a hydrophobic core surrounded by a hydrophilic corona. FNP produces nanoparticles with very high loading levels of nonionic hydrophobic APIs. However, hydrophobic compounds with ionizable groups are not as efficiently incorporated. To overcome this, ion pairing agents (IPs) can be incorporated into the FNP formulation to produce highly hydrophobic drug salts that efficiently precipitate during mixing. We demonstrate the encapsulation of the PI3K inhibitor, LY294002, within poly(ethylene glycol)-b-poly(D,L lactic acid) nanoparticles. We investigated how incorporating two hydrophobic IPs (palmitic acid (PA) and hexadecylphosphonic acid (HDPA)) during the FNP process affected the LY294002 loading and size of the resulting nanoparticles. The effect of organic solvent choice on the synthesis process was also examined. While the presence of either hydrophobic IP effectively increased the encapsulation of LY294002 during FNP, HDPA resulted in well-defined colloidally stable particles, while the PA resulted in ill-defined aggregates. The incorporation of hydrophobic IPs with FNP opens the door for the intravenous administration of APIs that were previously deemed unusable due to their hydrophobic nature. Full article

For many years, researchers have been making efforts to find a manufacturing technique, as well as a drug delivery system, that will allow for oral delivery of biopharmaceuticals to their target site of action without impairing their biological activity. Due to the positive in vivo outcomes of this formulation strategy, self-emulsifying drug delivery systems (SEDDSs) have been intensively studied in the last few years as a way of overcoming the different challenges associated with the oral delivery of macromolecules. The purpose of the present study was to examine the possibility of developing solid SEDDSs as potential carriers for the oral delivery of lysozyme (LYS) using the Quality by Design (QbD) concept. LYS was successfully ion paired with anionic surfactant, sodium dodecyl sulphate (SDS), and this complex was incorporated into a previously developed and optimized liquid SEDDS formulation comprising medium-chain triglycerides, polysorbate 80, and PEG 400. The final formulation of a liquid SEDDS carrying the LYS:SDS complex showed satisfactory in vitro characteristics as well as self-emulsifying properties (droplet size: 13.02 nm, PDI: 0.245, and zeta potential: −4.85 mV). The obtained nanoemulsions were robust to dilution in the different media and highly stable after 7 days, with a minor increase in droplet size (13.84 nm) and constant negative zeta potential (−0.49 mV). An optimized liquid SEDDS loaded with the LYS:SDS complex was further solidified into powders by adsorption onto a chosen solid carrier, followed by direct compression into self-emulsifying tablets. Solid SEDDS formulations also exhibited acceptable in vitro characteristics, while LYS preserved its therapeutic activity in all phases of the development process. On the basis of the results gathered, loading the hydrophobic ion pairs of therapeutic proteins and peptides to solid SEDDS may serve as a potential method for delivering biopharmaceuticals orally. Full article

Ionic perturbation of water has important implications in various chemical, biological and environmental processes. Previous studies revealed the structural and dynamical perturbation of water in the presence of ions, mainly with concentrated electrolyte solutions having significant interionic interactions. These investigations highlighted the need of selective extraction of the hydration shell water from a dilute electrolyte solution that is largely free from interionic interactions. Double-difference infrared (DDIR) and Raman multivariate curve resolution (Raman-MCR), as well as MD simulation, provided valuable insight in this direction, suggesting that the perturbed water mainly resides in the immediate vicinity of the ion, called the hydration shell. Recently, we have introduced Raman difference spectroscopy with simultaneous curve fitting (Raman-DS-SCF) analysis that can quantitatively extract the vibrational response of the perturbed water pertaining to the hydration shell of fully hydrated ions/solute. The DS-SCF analysis revealed novel hydrogen-bond (H-bond) structural features of hydration water, such as the existence of extremely weakly interacting water–OH (ν_max ~ 3600 cm⁻¹) in the hydration shell of high-charge-density metal ions (Mg²⁺, Dy³⁺). In addition, Raman-DS-SCF retrieves the vibrational response of the shared water in the water–shared-ion pair (WSIP), which is different from the hydration shell water of either the interacting cation and anion. Herein, we discuss the perturbation of water H-bonding in the immediate vicinity of cation, anion, zwitterion and hydrophobes and also the inter-ionic interactions, with a focus on the recent results from our laboratory using Raman-DS-SCF spectroscopy. Full article