Preparation and Characterization of Disulfiram and Beta Cyclodextrin Inclusion Complexes for Potential Application in the Treatment of SARS-CoV-2 via Nebulization

Disulfiram (DS), known as an anti-alcoholism drug, has shown a potent antiviral activity. Still, the potential clinical application of DS is limited by its low water solubility and rapid metabolism. Cyclodextrins (CDs) have been widely used to improve the solubility of drugs in water. In this study, five concentrations of hydroxypropyl β-cyclodextrin (HP) and sulfobutyl ether β-cyclodextrin (SBE) were used to form inclusion complexes of DS for enhanced solubility. Solutions were freeze-dried, and the interaction between DS and CD was characterized using differential scanning calorimetry (DSC), thermogravimetric analysis (TGA), and Fourier transform infrared spectroscopy (FTIR). In addition, the nebulization properties of the DS–CD solutions were studied. The aqueous solubility of DS increased significantly when loaded to either of both CDs. The phase solubility of both complexes was a linear function of the CD concentration (A_L type). Furthermore, physicochemical characterization studies showed a potent inclusion of the drug in the CD–DS complexes. Aerosolization studies demonstrated that these formulations are suitable for inhalation. Overall, the CD inclusion complexes have great potential for the enhancement of DS solubility. However, further studies are needed to assess the efficacy of DS–CD inclusion complexes against SARS-CoV-2 via nebulization.     

Enhancement of Dissolving Capacity and Reducing Gastric Mucosa Irritation by Complex Formation of Resibufogenin with β-Cyclodextrin or 2-Hydroxypropyl-β-cyclodextrin

Resibufogenin (RBG) is a natural medicinal ingredient with promising cardiac protection and antitumor activity. However, poor solubility and severe gastric mucosa irritation restrict its application in the pharmaceutical field. In this study, the inclusion complex of RBG with β-cyclodextrin (β-CD) and 2-hydroxypropyl-β-cyclodextrin (HP-β-CD) was prepared using the co-evaporation method, and the molar ratio of RBG to CD was determined to be approximately 1:2 by continuous variation plot for both CDs. The formation of inclusion complexes between RBG and each CD (RBG/β-CD and RBG/HP-β-CD) was evaluated by phase solubility study, Fourier transform infrared spectroscopy, and thin-layer chromatography. Powder X-ray diffraction and differential scanning calorimetry confirmed drug amorphization and encapsulation in the molecular cage for both CDs. Moreover, the inclusion complexes’ morphologies were observed using scanning electron microscopy. The dissolution rate of the inclusion complexes was markedly improved compared to that of RBG, and the complexes retained their antitumor activity, as shown in the in vitro cytotoxicity assay on a human lung adenocarcinoma cancer (A549) cell line. Moreover, less gastric mucosal irritation was observed for the inclusion complex. Thus, the inclusion complex should be considered a promising strategy for the delivery of poorly water-soluble anticancer agents, such as RBG.     

Encapsulation of Risperidone by Methylated β-Cyclodextrins: Physicochemical and Molecular Modeling Studies

Risperidone (RSP) is an atypical antipsychotic drug which acts as a potent antagonist of serotonin-2 (5TH2) and dopamine-2 (D2) receptors in the brain; it is used to treat schizophrenia, behavioral and psychological symptoms of dementia and irritability associated with autism. It is a poorly water soluble benzoxazole derivative with high lipophilicity. Supramolecular adducts between drug substance and two methylated β-cyclodextrins, namely heptakis(2,6-di-O-methyl)-β-cyclodextrin (DM-β-CD) and heptakis(2,3,6-tri-O-methyl)-β-cyclodextrin (TM-β-CD) were obtained in order to enhance RSP solubility and improve its biopharmaceutical profile. The inclusion complexes were evaluated by means of thermoanalytical methods (TG—thermogravimetry/DTG—derivative thermogravimetry/HF—heat flow), powder X-ray diffractometry (PXRD), universal-attenuated total reflectance Fourier transform infrared (UATR-FTIR), UV spectroscopy and saturation solubility studies. Job’s method was employed for the determination of the stoichiometry of the inclusion complexes, which was found to be 2:1 for both guest–host systems. Molecular modeling studies were carried out for an in-depth characterization of the interaction between drug substance and cyclodextrins (CDs). The physicochemical properties of the supramolecular systems differ from those of RSP, demonstrating the inclusion complex formation between drug and CDs. The RSP solubility was enhanced as a result of drug encapsulation in the CDs cavity, the higher increase being obtained with DM-β-CD as host; the guest–host system RSP/DM-β-CD can thus be a starting point for further research in developing new formulations containing RSP, with enhanced bioavailability.     

N,N-Diethyl-3-toluamide Formulation Based on Ethanol Containing 0.1% 2-Hydroxypropyl-β-cyclodextrin Attenuates the Drug’s Skin Penetration and Prolongs the Repellent Effect without Stickiness

N,N-diethyl-3-toluamide (DEET) is one of the most widely used insect repellents in the world. It was reported that a solution containing 6–30% cyclodextrin (CD) as a solvent instead of ethanol (EtOH) provided an enhancement of the repellent action time duration of the DEET formulation, although the high-dose CD caused stickiness. In order to overcome this shortcoming, we attempted to prepare a 10% DEET formulation using EtOH containing low-dose CDs (β-CD, 2-hydroxypropyl-β-CD (HPβCD), methyl-β-CD, and sulfobutylether-β-CD) as solvents (DEET/EtOH/CD formulations). We determined the CD concentration to be 0.1% in the DEET/EtOH/CD formulations, since the stickiness of 0.1% CDs was not felt (approximately 8 × 10⁻³ N). The DEET residue on the skin superficial layers was prolonged, and the drug penetration into the skin tissue was decreased by the addition of 0.1% CD. In particular, the retention time and attenuated penetration of DEET on the rat skin treated with the DEET/EtOH/HPβCD formulation was significantly higher in comparison with that of the DEET/EtOH formulation without CD. Moreover, the repellent effect of DEET was more sustained by the addition of 0.1% HPβCD in the study using Aedes albopictus. In conclusion, we found that the DEET/EtOH/HPβCD formulations reduced the skin penetration of DEET and prolonged the repellent action without stickiness.     

Chiral Discrimination of Mexiletine Enantiomers by Capillary Electrophoresis Using Cyclodextrins as Chiral Selectors and Experimental Design Method Optimization

Mexiletine (MXL) is a class IB antiarrhythmic agent, acting as a non-selective voltage-gated sodium channel blocker, used in therapy as a racemic mixture R,S-MXL hydrochloride. The aim of the current study was the development of a new, fast, and efficient method for the chiral separation of MXL enantiomers using capillary electrophoresis (CE) and cyclodextrins (CDs) as chiral selectors (CSs). After an initial CS screening, using several neutral and charged CDs, at four pH levels, heptakis-2,3,6-tri-O-methyl-β-CD (TM-β-CD), a neutral derivatized CD, was chosen as the optimum CS for the enantioseparation. For method optimization, an initial screening fractional factorial design was applied to identify the most significant parameters, followed by a face-centered central composite design to establish the optimal separation conditions. The best results were obtained by applying the following optimized electrophoretic conditions: 60 mM phosphate buffer, pH 5.0, 50 mM TM-β-CD, temperature 20 °C, applied voltage 30 kV, hydrodynamic injection 50 mbar/s. MXL enantiomers were baseline separated with a resolution of 1.52 during a migration time of under 5 min; S-MXL was the first migrating enantiomer. The method’s analytical performance was verified in terms of precision, linearity, accuracy, and robustness (applying a Plackett–Burman design). The developed method was applied for the determination of MXL enantiomers in pharmaceuticals. A computer modeling of the MXL-CD complexes was applied to characterize host–guest chiral recognition.     

The Effect of α-, β- and γ-Cyclodextrin on Wheat Dough and Bread Properties

Cyclodextrins (CDs) are cyclic oligosaccharides that have found widespread application in numerous fields. CDs have revealed a number of various health benefits, making them potentially useful food supplements and nutraceuticals. In this study, the impact of α-, β-, and γ-CD at different concentrations (up to 8% of the flour weight) on the wheat dough and bread properties were investigated. The impact on dough properties was assessed by alveograph analysis, and it was found that especially β-CD affected the viscoelastic properties. This behavior correlates well with a direct interaction of the CDs with the proteins of the gluten network. The impact on bread volume and bread staling was also assessed. The bread volume was in general not significantly affected by the addition of up to 4% CD, except for 4% α-CD, which slightly increased the bread volume. Larger concentrations of CDs lead to decreasing bread volumes. Bread staling was investigated by texture analysis and low field nuclear magnetic resonance spectroscopy (LF-NMR) measurements, and no effect of the addition of CDs on the staling was observed. Up to 4% CD can, therefore, be added to wheat bread with only minor effects on the dough and bread properties.     

Polysorbates versus Hydroxypropyl Beta-Cyclodextrin (HPβCD): Comparative Study on Excipient Stability and Stabilization Benefits on Monoclonal Antibodies

Polysorbates (PS 20 and PS 80) are the most widely used surfactants in biopharmaceutical formulations to protect proteins from denaturation, aggregation, and surface adsorption. To date, around 70% of marketed therapeutic antibodies contain either PS 20 or PS 80 in their formulations. However, polysorbates are chemically diverse mixtures, which are prone to degradation by oxidation and hydrolysis to produce peroxides and fatty acids, which, in turn, induce protein oxidation, aggregation, and insoluble particle formation. These will negatively impact protein quality and stability. Thus, polysorbate degradation has emerged as one of the major challenges in the development and commercialization of therapeutic protein products. KLEPTOSE^® HPβCD (hydroxypropyl beta-cyclodextrin), a new multifunctional excipient, has been shown to provide protein stabilization functions in biopharmaceutical downstream processes and in their final formulations. This study aims to evaluate HPβCD, a new molecule of its class, against polysorbates as a stabilizer in biologics formulations. In this study, the chemical stability of KLEPTOSE^® HPβCDs is compared with polysorbates (20 and 80) under various stress conditions. When subjected to heat stress, HPβCDs show little change in product recovery (90.7–100.7% recovery for different HPβCDs), while polysorbates 20 and 80 show significant degradation, with only 11.5% and 7.3% undegraded product remaining, respectively. When subjected to other chemical stressors, namely, autoclave, light, and oxidative stresses, HPβCD remains almost stable, while polysorbates show more severe degradation, with 95.5% to 98.8% remaining for polysorbate 20 and 85.5% to 97.4% remaining for polysorbate 80. Further, profiling characterization and degradation analysis reveal that chemical structures of HPβCDs remain intact, while polysorbates undergo significant hydrolytic degradation and oxidation. Lastly, the physicochemical stability of monoclonal antibodies in formulations is investigated. When subjected to light stress, adalimumab, as a model mAb, formulated in the presence of HPβCD, shows a significant decrease in protein aggregation, and superior monomer and total protein recovery compared to PS 80-containing formulations. HPβCD also reduces both agitation and thermal stress-induced protein aggregation and prevents subvisible particle formation compared to PS 80.     

Cyclodextrins Initiated Ring-Opening Polymerization of Lactide Using 4-Dimethylaminopyridine (DMAP) as Catalyst: Study of DMAP/β-CD Inclusion Complex and Access to New Structures

Cyclodextrins (CDs) are cyclic oligosaccharides used in many fields. Grafting polymers onto CDs enables new structures and applications to be obtained. Polylactide (PLA) is a biobased, biocompatible aliphatic polyester that can be grafted onto CDs by -OH-initiated ring-opening polymerization. Using 4-dimethylaminopyridine (DMAP) as an organocatalyst, a quantitative functionalization is reached on native α-, β-, γ- and 2,3-dimethyl- β-cyclodextrins. Narrow molecular weight distributions are obtained with the native CDs (dispersity < 1.1). The DMAP/β-CD combination is used as a case study, and the formation of an inclusion complex (1/1) is shown for the first time in the literature, which is fully characterized by NMR. The inclusion of DMAP into the cavity occurs via the secondary rim of the β-CD and the association constant (K_a) is estimated to be 88.2 M⁻¹. Its use as an initiator for ring-opening polymerization leads to a partial functionalization efficiency, and thus a more hydrophilic β-CD-PLA conjugate than that obtained starting from native β-CD. Polymerization results including also the use of the adamantane/β-CD inclusion complex as an initiator suggest that inclusion of the DMAP catalyst into the CD may not occur during polymerization reactions. Rac-lactide does not form an inclusion complex with β-CD.     

Synthesis of Fluorescent Carbon Dots and Their Application in Ascorbic Acid Detection

Water-soluble fluorescent carbon dots (CDs) were synthesized by a hydrothermal method using citric acid as the carbon source and ethylenediamine as the nitrogen source. The repeated and scale-up synthetic experiments were carried out to explore the feasibility of macroscopic preparation of CDs. The CDs/Fe³⁺ composite was prepared by the interaction of the CDs solution and Fe³⁺ solution. The optical properties, pH dependence and stability behavior of CDs or the CDs/Fe³⁺ composite were studied by ultraviolet spectroscopy and fluorescence spectroscopy. Following the principles of fluorescence quenching after the addition of Fe³⁺ and then the fluorescence recovery after the addition of asorbic acid, the fluorescence intensity of the carbon dots was measured at λex = 360 nm, λem = 460 nm. The content of ascorbic acid was calculated by quantitative analysis of the changing fluorescence intensity. The CDs/Fe³⁺ composite was applied to the determination of different active molecules, and it was found that the composite had specific recognition of ascorbic acid and showed an excellent linear relationship in 5.0–350.0 μmol·L⁻¹. Moreover, the detection limit was 3.11 μmol·L⁻¹. Satisfactory results were achieved when the method was applied to the ascorbic acid determination in jujube fruit. The fluorescent carbon dots composites prepared in this study may have broad application prospects in a rapid, sensitive and trace determination of ascorbic acid content during food processing.     

Preparation and Properties of Cyclodextrin Inclusion Complexes of Hyperoside

In order to improve the aqueous solubility and enhance the bioavailability of Hyperoside (Hyp), three inclusion complexes (ICs) of Hyp with 2-hydroxypropyl-β-cyclodextrin (2H-β-CD), β-cyclodextrin (β-CD), and methyl-β-cyclodextrin (M-β-CD) were prepared using the ultrasonic method. The characterization of the inclusion complexes (ICs) was achieved using Fourier-transform infrared spectroscopy (FTIR), scanning electronic microscopy (SEM), X-ray powder diffraction (XRPD), thin-layer chromatography (TLC), and ¹H nuclear magnetic resonance (¹H NMR). The effects of the ICs on the solubility and antioxidant activity of Hyp were investigated. A Job’s plot revealed that the Hyp formed ICs with three kinds of cyclodextrin (CD), all at a 1:1 stoichiometric ratio. The FTIR, SEM, XRPD, TLC, and ¹H NMR results confirmed the formation of inclusion complexes. The water solubility of the IC of Hyp with 2-hydroxypropyl-β-cyclodextrin was enhanced 9-fold compared to the solubility of the original Hyp. The antioxidant activity tests showed that the inclusion complexes had higher antioxidant activities compared to free Hyp in vitro and the H₂O₂–RAW264.7 cell model. Therefore, encapsulation with CDs can not only improve Hyp’s water solubility but can also enhance its biological activity, which provides useful information for the potential application of complexation with Hyp in a clinical context.     

On the Interactions of Melatonin/β-Cyclodextrin Inclusion Complex: A Novel Approach Combining Efficient Semiempirical Extended Tight-Binding (xTB) Results with Ab Initio Methods

Melatonin (MT) is a molecule of paramount importance in all living organisms, due to its presence in many biological activities, such as circadian (sleep–wake cycle) and seasonal rhythms (reproduction, fattening, molting, etc.). Unfortunately, it suffers from poor solubility and, to be used as a drug, an appropriate transport vehicle has to be developed, in order to optimize its release in the human tissues. As a possible drug-delivery system, β-cyclodextrin (βCD) represents a promising scaffold which can encapsulate the melatonin, releasing when needed. In this work, we present a computational study supported by experimental IR spectra on inclusion MT/βCD complexes. The aim is to provide a robust, accurate and, at the same time, low-cost methodology to investigate these inclusion complexes both with static and dynamic simulations, in order to study the main actors that drive the interactions of melatonin with β-cyclodextrin and, therefore, to understand its release mechanism.     

Fluorescence properties of (R)- and (S)-[1,1′-binaphthalene]-2,2′-diols solutions and their complexes with cyclodextrins in aqueous medium

Steady-state and time-resolved fluorescence techniques were used to study (R)- and (S)-[1,1′-binaphthalene]-2,2′-diol (1,1′-binaphthol or BINOL) dilute solutions of different polarity solvents, as well as their inclusion complexes with α- and βcyclodextrins (CDs) in water. BINOLs in dilute water solutions exhibited a surprisingly high fluorescence anisotropy that was explained as being due to the formation of fairly large order π–π stacking aggregates in aqueous polar media. Stoichiometries, formation constants and the changes of enthalpy and entropy upon inclusion were also obtained by measuring the variation of the fluorescence intensity with [CD] and temperature. Results agree with the formation of 1:1 stoichiometry complexes, but the association constants are rather low and very similar for both enantiomers. Molecular mechanic calculations in the presence of water were employed to study the formation of BINOL complexes with both α- and βCDs. For the most stable structures of any of the complexes only a small portion of the guests, in agreement with thermodynamics parameters and quenching experiments, penetrates inside the CD cavities. Driving forces for 1:1 inclusion processes may be dominated by non-bonded van der Waals host:guest interactions. The low guest:host binding constants and poor enantioselectivity of α- and βCDs for BINOLS may be a consequence of the BINOL aggregation in water.     

Cyclodextrin Complexed Lipid Nanoparticles of Irbesartan for Oral Applications: Design,Development, and In Vitro Characterization

Irbesartan (IR) is an angiotensin II receptor antagonist drug with antihypertensive activity. IR bioavailability is limited due to poor solubility and first-pass metabolism. The current investigation aimed to design, develop, and characterize the cyclodextrin(s) (CD) complexed IR (IR-CD) loaded solid lipid nanoparticles (IR-CD-SLNs) for enhanced solubility, sustained release behavior, and subsequently improved bioavailability through oral administration. Based on phase solubility studies, solid complexes were prepared by the coacervation followed by lyophilization method and characterized for drug content, inclusion efficiency, solubility, and in vitro dissolution. IR-CD inclusion complexes demonstrated enhancement of solubility and dissolution rate of IR. However, the dissolution efficiency was significantly increased with hydroxypropyl-βCD (HP-βCD) inclusion complex than beta-CD (βCD). SLNs were obtained by hot homogenization coupled with the ultrasonication method with IR/HP-βCD inclusion complex loaded into Dynasan 112 and glycerol monostearate (GMS). SLNs were evaluated for physicochemical characteristics, in vitro release, differential scanning calorimetry (DSC), powder X-ray diffractometry (PXRD), and physical stability at room temperature for two months. The optimized SLNs formulation showed particle size, polydispersity index, zeta potential, assay, and entrapment efficiency of 257.6 ± 5.1 nm, 0.21 ± 0.03, −30.5 ± 4.1 mV, 99.8 ± 2.5, and 93.7 ± 2.5%, respectively. IR-CD-SLN and IR-SLN dispersions showed sustained release of IR compared to the IR-CD inclusion complexes. DSC results complimented PXRD results by the absence of IR endothermic peak. Optimized IR-CD complex, IR-SLN, and IR-CD-SLN formulations were stable for two months at room temperature. Thus, the current IR oral formulation may exhibit improved oral bioavailability and prolonged antihypertensive activity, which may improve therapeutic outcomes in the treatment of hypertension and heart failure.     

Single Isomer N-Heterocyclic Cyclodextrin Derivatives as Chiral Selectors in Capillary Electrophoresis

In order to better understand the chiral recognition mechanisms of positively charged cyclodextrin (CD) derivatives, the synthesis, the pK_a determination by ¹H nuclear magnetic resonance (NMR)-pH titration and a comparative chiral capillary electrophoretic (CE) study were performed with two series of mono-substituted cationic single isomer CDs. The first series of selectors were mono-(6-N-pyrrolidine-6-deoxy)-β-CD (PYR-β-CD), mono-(6-N-piperidine-6-deoxy)-β-CD (PIP-β-CD), mono-(6-N-morpholine-6-deoxy)-β-CD (MO-β-CD) and mono-(6-N-piperazine-6-deoxy)-β-CD (PIPA-β-CD), carrying a pH-adjustable moiety at the narrower rim of the cavity, while the second set represented by their quaternarized, permanently cationic counterparts: mono-(6-N-(N-methyl-pyrrolidine)-6-deoxy)-β-CD (MePYR-β-CD), mono-(6-N-(N-methyl-piperidine)-6-deoxy)-β-CD (MePIP-β-CD), mono-(6-N-(N-methyl-morpholine)-6-deoxy)-β-CD (MeMO-β-CD) and mono-(6-N-(4,4-N,N-dimethyl-piperazine)-β-CD (diMePIPA-β-CD). Based on pH-dependent and selector concentration-dependent comparative studies of these single isomer N-heterocyclic CDs presented herein, it can be concluded that all CDs could successfully be applied as chiral selectors for the enantiodiscrimination of several negatively charged and zwitterionic model racemates. The substituent-dependent enantiomer migration order reversal of dansylated-valine using PIP-β-CD contrary to PYP-β-CD, MO-β-CD and PIPA-β-CD was also studied by ¹H- and 2D ROESY NMR experiments.     

Complexation of Ketoconazole by Native and Modified Cyclodextrins

Complexation of ketoconazole (KET), a broad-spectrum antifungal drug, with β- and γ-cyclodextrins (CDs), heptakis (2,6-di-O-methyl)-β-CD (2,6-DM-β-CD), heptakis (2,3,6-tri-O-methyl)-β-CD (TM-β-CD), 2-hydroxypropyl-β-CD (2HP-β-CD) and carboxymethyl-β-CD (CM-β-CD) was studied. The stability constants were determined by the solubility method at pH = 6 and for 2,6-DM-β-CD and CM-β-CD at pH = 5. At pH = 6, the stability constants increased in the order: TM-β-D < γ-CD < 2HP-β-CD < β-CD < CM-β-CD < 2,6-DM-β-CD. At pH = 5, due to the increased ionization of KET, the stability constant with CM-β-CD increased and with 2,6-DM-β-CD decreased. For complexes of KET with 2HP-β-CD and 2,6-DM-β-CD, the thermodynamic parameters of complexation were determined from the temperature dependence of the corresponding stability constants. For β–γ and TM-β-CD complexes, calculations using HyperChem 6 software by the Amber force field were carried out to gain some insight into the host–guest geometry.     

Comparative Interaction Studies of Quercetin with 2-Hydroxyl-propyl-β-cyclodextrin and 2,6-Methylated-β-cyclodextrin

Quercetin (QUE) is a well-known natural product that can exert beneficial properties on human health. However, due to its low solubility its bioavailability is limited. In the present study, we examine whether its formulation with two cyclodextrins (CDs) may enhance its pharmacological profile. Comparative interaction studies of quercetin with 2-hydroxyl-propyl-β-cyclodextrin (2HP-β-CD) and 2,6-methylated cyclodextrin (2,6Me-β-CD) were performed using NMR spectroscopy, DFT calculations, and in silico molecular dynamics (MD) simulations. Using T1 relaxation experiments and 2D DOSY it was illustrated that both cyclodextrin vehicles can host quercetin. Quantum mechanical calculations showed the formation of hydrogen bonds between QUE with 2HP-β-CD and 2,6Μe-β-CD. Six hydrogen bonds are formed ranging between 2 to 2.8 Å with 2HP-β-CD and four hydrogen bonds within 2.8 Å with 2,6Μe-β-CD. Calculations of absolute binding free energies show that quercetin binds favorably to both 2,6Me-β-CD and 2HP-β-CD. MM/GBSA results show equally favorable binding of quercetin in the two CDs. Fluorescence spectroscopy shows moderate binding of quercetin in 2HP-β-CD (520 M⁻¹) and 2,6Me-β-CD (770 M⁻¹). Thus, we propose that both formulations (2HP-β-CD:quercetin, 2,6Me-β-CD:quercetin) could be further explored and exploited as small molecule carriers in biological studies.     

Synthesis of Mono-Amino Substituted γ-CD: Host–Guest Complexation and In Vitro Cytotoxicity Investigation

Cyclodextrins (CDs) are cyclic oligosaccharides which can trap hydrophobic molecules and improve their chemical, physical, and biological properties. γ-CD showed the highest aqueous solubility with the largest cavity diameter among other CD types. The current study describes a direct and easy method for nucleophilic mono-aminos to be substituted with γ-CD and tested for their ability to host the guest curcumin (CUR) as a hydrophobic drug model. The mass spectrometry and NMR analyses showed the successful synthesis of three amino-modified γ-CDs: mono-6-amino-6-deoxy-cyclodextrine (γ-CD-NH₂), mono-6-deoxy-6-ethanolamine-γ-cyclodextrine (γ-CD-NHCH₂CH₂OH), and mono-6-deoxy-6-aminoethylamino)-γ-cyclodextrin (γ-CD-NHCH₂CH₂NH₂). These three amino-modified γ-CDs were proven to be able to host CUR as native γ-CDs with formation constants equal to 6.70 ± 1.02, 5.85 ± 0.80, and 8.98 ± 0.90 mM⁻¹, respectively. Moreover, these amino-modified γ-CDs showed no significant toxicity against human dermal fibroblast cells. In conclusion, the current work describes a mono-substitution of amino-modified γ-CDs that can still host guests and showed low toxicity in human dermal fibroblasts cells. Therefore, the amino-modified γ-CDs can be used as a carrier host and be conjugated with a wide range of molecules for different biomedical applications, especially for active loading methods.     

Development of a Microsphere-Based Immunoassay Authenticating A2 Milk and Species Purity in the Milk Production Chain

Processed milk and milk products produced from bovine milk, commonly contain β-casein A1 (βCA1) and β-casein A2 (βCA2). Since the presence of βCA1 is linked to milk intolerance and digestion problems, A2A2 milk, which only contains βCA2, is proposed as a healthier alternative. To support this health claim, the purity of A2A2-milk has to be guaranteed. In the presented study, a multiplex immunoassay, able to distinguish between βCA2 and βCA1, was developed and real-life applicability was shown on raw milk samples from genotyped A1A1, A1A2 and A2A2 cows. Because of its ability to discriminate between βCA2 and βCA1, this newly developed method was able to detect the addition of common bovine A1A2 milk to A2A2 milk, as low as 1%. Besides the detection of A2A2 milk purity, the developed assay can also be implemented as a rapid phenotyping method at dairy farms to replace the more invasive DNA-based screening. Additionally, the developed method was capable of detecting the addition of common bovine milk up to 1% in sheep, goat, buffalo, horse and donkey milk, which conforms to EU recommendations. In conclusion, a newly developed multiplex method capable of reliably detecting the dilution of A2A2 milk of multiple species, with common bovine milk up to 1%, is presented.     

Encapsulation of α-Pinene in Delivery Systems Based on Liposomes and Cyclodextrins

The essential oil component α-pinene has multiple biological activities. However, its application is limited owing to its volatility, low aqueous solubility, and chemical instability. For the aim of improving its physicochemical properties, α-pinene was encapsulated in conventional liposomes (CLs) and drug-in-cyclodextrin-in-liposomes (DCLs). Hydroxypropyl-β-cyclodextrin/α-pinene (HP-β-CD/α-pinene) inclusion complexes were prepared in aqueous solution, and the optimal solubilization of α-pinene occurred at HP-β-CD:α-pinene molar ratio of 7.5:1. The ethanol-injection method was applied to produce different formulations using saturated (Phospholipon 90H) or unsaturated (Lipoid S100) phospholipids in combination with cholesterol. The size, the phospholipid and cholesterol incorporation rates, the encapsulation efficiency (EE), and the loading rate (LR) of α-pinene were determined, and the storage stability of liposomes was assessed. The results showed that α-pinene was efficiently entrapped in CLs and DCLs with high EE values. Moreover, Lipoid S100 CLs displayed the highest LR (22.9 ± 2.2%) of α-pinene compared to the other formulations. Both carrier systems HP-β-CD/α-pinene inclusion complex and Lipoid S100 CLs presented a gradual release of α-pinene. Furthermore, the DPPH radical scavenging activity of α-pinene was maintained upon encapsulation in Lipoid S100 CLs. Finally, it was found that all formulations were stable after three months of storage at 4 °C.     

The Effects of Charged Amino Acid Side-Chain Length on Diagonal Cross-Strand Interactions between Carboxylate- and Ammonium-Containing Residues in a β-Hairpin

The β-sheet is one of the common protein secondary structures, and the aberrant aggregation of β-sheets is implicated in various neurodegenerative diseases. Cross-strand interactions are an important determinant of β-sheet stability. Accordingly, both diagonal and lateral cross-strand interactions have been studied. Surprisingly, diagonal cross-strand ion-pairing interactions have yet to be investigated. Herein, we present a systematic study on the effects of charged amino acid side-chain length on a diagonal ion-pairing interaction between carboxylate- and ammonium-containing residues in a β-hairpin. To this end, 2D-NMR was used to investigate the conformation of the peptides. The fraction folded population and the folding free energy were derived from the chemical shift data. The fraction folded population for these peptides with potential diagonal ion pairs was mostly lower compared to the corresponding peptide with a potential lateral ion pair. The diagonal ion-pairing interaction energy was derived using double mutant cycle analysis. The Asp2-Dab9 (Asp: one methylene; Dab: two methylenes) interaction was the most stabilizing (−0.79 ± 0.14 kcal/mol), most likely representing an optimal balance between the entropic penalty to enable the ion-pairing interaction and the number of side-chain conformations that can accommodate the interaction. These results should be useful for designing β-sheet containing molecular entities for various applications.