Structural analysis of PEGylated nanoemulsions using EPR spectroscopy – the impact of an active compound incorporated in stabilizing layer

INTRODUCTION Nanoemulsions (NEs) offer a flexible platform for drug delivery via several administration routes. Rapid plasma clearance brought on by interactions with plasma proteins and the activation of the mononuclear phagocytic system is the greatest challenge NEs face after parenteral administration. PEGylation, or adding PEGylated phospholipids to the stabilizing layer of the NEs, is one method for ensuring that droplets circulate for a longer period of time. It is crucial to select the optimum concentration of the PEGylated in order to maintain the necessary physicochemical properties of NEs while providing appropriate surface coverage with the PEG chains. Curcumin is a model active that has been found to be localized in the stabilizing layer of NEs (1-3) and offers a wide range of potential health benefits, but due to its short plasma half-life, new strategies for enhancing bioavailability are required. The purpose of this study is to investigate the effects of various PEGylated phospholipid (PEG2000-DSPE) concentrations on the structural properties of NEs with an active placed in the stabilizing layer. PREPARATION OF NANOEMULSIONS All the NEs were prepared using the high pressure homogenization technique. The aqueous phase (glycerol, polysorbate 80, sodium oleate and highly purified water) was added to the oil phase (soybean oil, medium chain triglycerides, soybean lecithin, buthylhydroxytoluene, curcumin and 0.1%/0.3%/0.6% PEG2000-DSPE) and mixed at 11000rpm for 1 min on rotor stator homogenizer (IKA Ultra-Turrax T25 digital, IKA-Werke GmbH & Co. KG, Staufen, Germany), and then further processed at 800 bar for 10 discontinued cycles (EmulsiFlex-C3, Avestin Inc., Ottawa,ON, Canada) to obtain CS21, CS23 and CS26 formulations. NANOEMULSION DROPLET SIZE The droplet size was assessed through the dynamic light scattering method and presented as mean droplet size (Z-ave) and polydispersity index (PDI), after diluting the NEs 1:500 (v/v) in highly purified water. ELECTRON PARAMAGNETIC RESONANCE (EPR) SPECTROSCOPY For this study three amphiphilic fatty acid derivatives labeled at different positions of the aliphatic chain (5-DSA, 12-DSA and 16-DSA) were used to probe the dynamics of the membrane at different depths. Stock solutions of the spin probes were prepared in absolute ethanol at 1mM concentration. Subsequently, 15 µl of the stock solutions were evaporated and then incubated with 1 ml of the NE sample in final concentration of 0.015 mM. The resulting spectra was analyzed in terms of rotational correlation time (τR), order parameter (S) and isotropic hyperfine constant (αN). RESULTS AND DISCUSION All of the formulations had average droplet sizes between 95 and 103 nm and PDI values under 0.25, which indicated that they were suitable for parenteral administration. The results of the EPR investigation showed that the stabilizing layer changed as the amount of PEGylated phospholipids increased, indicating that the PEGylation threshold has not yet been reached in the stabilizing layer. The EPR research also showed that the 5-DSA spin probe's spectra were significantly affected by the addition of various PEGylated phospholipid concentrations (Figure 1). This indicates that the portion of the stabilizing layer nearest to the aqueous phase was the one most affected by the increase in the PEGylated phospholipid concentration. Table 1 provides the calculated values for the spectrum parameters. The mobility of the spin-probe and the time it takes for the spin-probe to make a full rotation is reflected in the τR parameter, which was changed the most, compared to the other parameters, by the variations in the PEGylated phospholipid content. A formulation with the most rigid stabilizing layer had the largest τR values, which, in this instance, was the formulation with 0.1% PEG2000-DSPE. It's interesting to note that the addition of PEGylated phospholipid had the opposite effect of strengthening the stabilizing layer. Further PEG2000-DSPE addition appeared to result in nanoemulsions with a less rigid stabilizing layer, possibly indicating that larger concentrations of the PEGylating agent lead to interface destabilization. The interactions between the curcumin, a symmetrical molecule with two aromatic ring systems and a bent conformation located in the stabilizing layer and the extra stabilizer are likely responsible for this. The other two spin probes (12-DSA and 16-DSA) provide information about the stabilizing layer located closer to the oil core. Based on the data provided in Table 1 it can be inferred that the PEGylation mostly affects the stabilizing layer's areas closest to the aqueous interface, leaving the parts closer to the oil core largely not impacted. CONCLUSION This study demonstrates that one of the key elements in assessing how PEGylation affects the NE system is the active's localization. To pick the concentration of the PEGylated phospholipid that will offer the best surface coverage without compromising the integrity of the interface, additional considerations must be addressed in the event of an active situated in the stabilizing layer. In this instance, it may be hypothesized that the lowest PEG2000-DSPE concentration of 0.1%, CS21, will produce NEs that can slow down curcumin release the most compared to the other two formulations. Additionally, given that further addition of the PEGylated phospholipid causes the formation of less rigid stabilizing layer, further inquiries should be made to see the impact of these changes on the interactions with plasma proteins and biological fate of the droplets upon administration

Analyzing the impact of the oil phase selection and curcumin presence on the nanoemulsion stabilizing layer using electron paramagnetic resonance spectroscopy

ANALYZING THE IMPACT OF THE OIL PHASE SELECTION AND CURCUMIN PRESENCE ON THE NANOEMULSION STABILIZING LAYER USING ELECTRON PARAMAGNETIC RESONANCE SPECTROSCOPY Jelena Đoković1*, Sotiria Demisli2, Vassiliki Papadimitriou2, Aristotelis Xenakis2, Snežana Savić1 1University of Belgrade – Faculty of Pharmacy, Department of Pharmaceutical Technology and Cosmetology, Belgrade, Serbia 2National Hellenic Research Foundation – Institute of Chemical Biology, Athens, Greece *[email protected] The stabilizing layer of nanoemulsions impacts their stability and destiny upon in vivo administration (1). The aim of this work was to gain information about the dynamics of the surfactants’ monolayer when different oils (soybean / fish) were used, and obtain data regarding the localization of curcumin (2), an active compound with many potential health benefits, using electron paramagnetic resonance (EPR) spectroscopy. Formulations were analysed using EPR technique with three different spin probes: 5-, 12- and 16-doxyl stearic acid (DSA), to investigate membrane dynamics at different depths. The results indicated that the oil type played a crucial role, not only on the structure, but also in the localization of the bioactive compound. The addition of curcumin changed the rotational correlation time (τR) values, most notably for 5-DSA, both in soybean oil and fish oil nanoemulsions, indicating its localization in the stabilizing layer, but with opposite effects. In the soybean oil nanoemulsion the addition of curcumin increased spin probe mobility, with τR decreasing from 2.18±0.60 ns to 1.66±0.61 ns, indicating a less rigid stabilizing structure, while in the fish oil formulations it resulted in a more rigid structure reflected in τR increase from 1.19±0.10 ns to 2.96±0.81 ns and 1.63±0.13 ns to 2.27±0.19 ns, for 5-DSA and 12-DSA, respectively. This study concluded that the curcumin is located in the stabilizing layer of nanoemulsions, but its impact on stabilizing layer structure depended on the oil phase selection, with particular stabilizing effects on fish oil nanoemulsions. References 1. Nikolic, I. et al. Curcumin-loaded low‐energy nanoemulsions: Linking EPR spectroscopy‐ analysed microstructure and antioxidant potential with in vitro evaluated biological activity. J. Mol. Liq. 2020, 301, 112479. 2. Griffith, O.H. and Jost, P.C. Lipid Spin Labels in Biological Membrane. In Spin Labeling, Theory and Applications; Berliner, L.J., Eds.; Academic Press: New York, NY, USA, 1976; pp 454–484 Acknowledgements This research was funded by the MESDT, Republic of Serbia through Grant Agreement with University of Belgrade – Faculty of Pharmacy No: 451-03-68/2022-14/200161 and supported by the Science Fund of the Republic of Serbia, GRANT No 7749108, Neuroimmune aspects of mood, anxiety and cognitive effects of leads/drug candidates acting at GABAA and/or sigma‐2 receptors: In vitro/in vivo delineation by nano‐ and hiPSC‐based platform - NanoCellEmоCo

The Impact of the Oil Phase Selection on Physicochemical Properties, Long-Term Stability, In Vitro Performance and Injectability of Curcumin-Loaded PEGylated Nanoemulsions

A nanotechnology-based approach to drug delivery presents one of the biggest trends in biomedical science that can provide increased active concentration, bioavailability, and safety compared to conventional drug-delivery systems. Nanoemulsions stand out amongst other nanocarriers for being biodegradable, biocompatible, and relatively easy to manufacture. For improved drug-delivery properties, longer circulation for the nanoemulsion droplets should be provided, to allow the active to reach the target site. One of the strategies used for this purpose is PEGylation. The aim of this research was assessing the impact of the oil phase selection, soybean or fish oil mixtures with medium chain triglycerides, on the physicochemical characteristics and injectability of curcumin-loaded PEGylated nanoemulsions. Electron paramagnetic resonance spectroscopy demonstrated the structural impact of the oil phase on the stabilizing layer of nanoemulsions, with a more pronounced stabilizing effect of curcumin observed in the fish oil nanoemulsion compared to the soybean oil one. The design of the experiment study, employed to simultaneously assess the impact of the oil phase, different PEGylated phospholipids and their concentrations, as well as the presence of curcumin, showed that not only the investigated factors alone, but also their interactions, had a significant influence on the critical quality attributes of the PEGylated nanoemulsions. Detailed physicochemical characterization of the NEs found all formulations were appropriate for parenteral administration and remained stable during two years of storage, with the preserved antioxidant activity demonstrated by DPPH and FRAP assays. In vitro release studies showed a more pronounced release of curcumin from the fish oil NEs compared to that from the soybean oil ones. The innovative in vitro injectability assessment, designed to mimic intravenous application, proved that all formulations tested in selected experimental setting could be employed in prospective in vivo studies. Overall, the current study shows the importance of oil phase selection when formulating PEGylated nanoemulsion

Development and Study of Nanoemulsions and Nanoemulsion-Based Hydrogels for the Encapsulation of Lipophilic Compounds

Biocompatible nanoemulsions and nanoemulsion-based hydrogels were formulated for the encapsulation and delivery of vitamin D3 and curcumin. The aforementioned systems were structurally studied applying dynamic light scattering (DLS), electron paramagnetic resonance (EPR) spectroscopy and viscometry. In vitro studies were conducted using Franz diffusion cells to investigate the release of the bioactive compounds from the nanocarriers. The cytotoxicity of the nanoemulsions was investigated using the thiazolyl blue tetrazolium bromide (MTT) cell proliferation assay and RPMI 2650 nasal epithelial cells as in vitro model. DLS measurements showed that vitamin D3 and curcumin addition in the dispersed phase of the nanoemulsions caused an increase in the size of the oil droplets from 78.6 ± 0.2 nm to 83.6 ± 0.3 nm and from 78.6 ± 0.2 nm to 165.6 ± 1.0 nm, respectively. Loaded nanoemulsions, in both cases, were stable for 60 days of storage at 25 °C. EPR spectroscopy revealed participation of vitamin D3 and curcumin in the surfactants monolayer. In vitro release rates of both lipophilic compounds from the nanoemulsions were comparable to the corresponding ones from the nanoemulsion-based hydrogels. The developed o/w nanoemulsions did not exhibit cytotoxic effect up to the concentration threshold of 1 mg/mL in the cell culture medium

Ανάπτυξη νανοφορέων για την ενθυλάκωση κανναβινοειδών και άλλων βιοδραστικών ουσιών

The main goal of the present thesis was the development of novel biocompatible nanodispersions to be used as carriers for diverse lipophilic bioactive compounds. Τhe purpose of these systems was the effective delivery of the encapsulated substances while providing alternative routes of administration, other than the oral which is the most commonly used. Two different nanodispersions were developed namely, oil-in-water nanoemulsions and nanoemulsion-filled hydrogels. These systems were used as carriers for the encapsulation and delivery of various lipophilic substances with pharmacological interest.Initially, the structure of the developed systems was elucidated in order to reveal possible differences and determine the localization of the encapsulated compounds. It was also essential to investigate whether the nanoemulsions’ structure was affected after its incorporation into the hydrogel matrix. In order to obtain the structural characterization of the formulated nanocarriers Dynamic Light Scattering (DLS), Electron Paramagnetic Resonance Spectroscopy (EPR), Confocal Fluorescence Microscopy (CFM), Cryo-Electron Microscopy (Cryo-EM) and Small-angle X-ray Scattering (SAXS) were performed. The investigation was carried out for both systems in the absence and presence of bioactive compounds. Regarding the nanoemulsions, the structural study revealed that the localization of the encapsulated compounds was dependent on their structure and had an effect on the size of the nanodroplets and the surfactant monolayer. Nevertheless, no significant changes in the structure of the nanoemulsion were observed after its incorporation into the hydrogel.In order to evaluate the suitability of the formulated nanodispersions to act as delivery vehicles of bioactive compounds different in vitro methods were implemented for their biological evaluation. The cytotoxic effect of both nanocarriers in the absence and presence of the encapsulated molecules was assessed through a cell viability assay using the cell lines RPMI 2650 and WS1 as models of the human nasal epithelium and skin, respectively. Subsequently, in vitro permeation study through Franz difussions cells and tape stripping experiments were conducted indicating that the nanoemulsion-filled hydrogels could be more effective for the delivery of bioactives through the skin since it demonstrated increased penetration and improved release profiles compared to the bioactive released from the nanoemulsions.Consequently, the proposed nanodispersions could be promising candidates as carriers for the effective delivery of various substances of pharmacological interest through the skin providing increased efficacy as well as the possibility of alternative delivery routes.Βασικός στόχος της παρούσας διατριβής ήταν η ανάπτυξη βιοσυμβατών νανοδιασπορών ως φορέων χορήγησης λιπόφιλων βιοδραστικών ενώσεων. Συγκεκριμένα αναπτύχθηκαν νανογαλακτώματα ελαίου-σε-νερό και υδρογέλες με ενσωματωμένο νανογαλάκτωμα με στόχο την αποτελεσματική χορήγηση των ενθυλακωμένων ουσιών με φαρμακολογικό ενδιαφέρον. Αρχικά, διευκρινίστηκε η δομή των συστημάτων που αναπτύχθηκαν ώστε να εντοπιστούν πιθανές διαφορές μεταξύ τους και να προσδιοριστεί η θέση των βιοδραστικών ουσιών. Βασική ήταν επίσης η μελέτη της δομής των νανογαλακτωμάτων μετά την ενσωμάτωσή τους στην υδρογέλη. Για τον δομικό χαρακτηρισμό χρησιμοποιήθηκαν Δυναμική Σκέδαση Φωτός (DLS), Φασματοσκοπία Ηλεκτρονικού Παραμαγνητικού Συντονισμού (EPR), Συνεστιακή Μικροσκοπία Φθορισμού (CFM), Κρυογονική Ηλεκτρονική Μικροσκοπία (Cryo-EM) και Σκέδαση Ακτίνων Χ σε μικρές γωνίες (SAXS). Η έρευνα διεξήχθη και για τα δύο συστήματα απουσία και παρουσία βιοδραστικών ενώσεων. Η δομική μελέτη των νανογαλακτωμάτων αποκάλυψε ότι η δομή των ενθυλακωμένων ουσιών επηρέαζε τον προσανατολισμό τους μέσα στα νανοσταγονίδια, είχε επίδραση στο μέγεθος των νανοσταγονιδίων και στη μονοστιβάδα των επιφανειοενεργών. Ωστόσο, δεν παρατηρήθηκαν σημαντικές αλλαγές στη δομή του νανογαλακτώματος μετά την ενσωμάτωσή του στην υδρογέλη.Για την αξιολόγηση της καταλληλότητας των προτεινόμενων νανοδιασπορών να δρουν ως φορείς για τη χορήγηση βιοδραστικών ουσιών, εφαρμόστηκαν διαφορετικές μέθοδοι in vitro για τη βιολογική τους αποτίμηση. Η κυτταροτοξικότητα και των δύο νανοφορέων απουσία και παρουσία των ενθυλακωμένων μορίων αξιολογήθηκε μέσω μελέτης αναστολής της κυτταρικής βιωσιμότητας με χρήση των κυτταρικών σειρών RPMI 2650 και WS1 ως μοντέλα του ανθρώπινου ρινικού και δερματικού επιθηλίου αντίστοιχα. Μέσω πειραμάτων in vitro μελετήθηκε η διαπερατότητα της βιοδραστικής στο δέρμα με χρήση της κυψελίδων διάχυσης Franz και με την τεχνική tape stripping. Βρέθηκε ότι οι υδρογέλες γεμάτες με νανογαλάκτωμα θα μπορούσαν να είναι πιο αποτελεσματικές για την χορήγηση βιοδραστικών μέσω του δέρματος, καθώς τους παρείχε αυξημένη διαπερατότητα και βελτιωμένα προφίλ απελευθέρωσης σε σύγκριση με τα νανογαλακτώματα. Συνεπώς, οι προτεινόμενες νανοδιασπορές είναι κατάλληλες για την αποτελεσματική χορήγηση διάφορων ουσιών μέσω του δέρματος παρέχοντας αυξημένη αποτελεσματικότητα καθώς και δυνατότητα εναλλακτικών οδών χορήγησης

Encapsulation of cannabidiol in oil-in-water nanoemulsions and nanoemulsion-filled hydrogels: A structure and biological assessment study

Hypothesis: Lipophilic cannabidiol can be solubilized in oil-in water nanoemulsions, which can then be impregnated into chitosan hydrogels forming another colloidal system that will facilitate cannabidiol's release. The delivery from both systems was compared, alongside structural and biological studies, to clarify the effect of the two carriers' structure on the release and toxicity of the systems. Experiments: Oil-in-water nanoemulsions (NEs) and the respective nanoemulsion-filled chitosan hydrogels (NE/HGs) were formulated as carriers of cannabidiol (CBD). Size, polydispersity and stability of the NEs were evaluated and then membrane dynamics, shape and structure of both systems were investigated with EPR spin probing, SAXS and microscopy. Biocompatibility of the colloidal delivery systems was evaluated through cytotoxicity tests over normal human skin fibroblasts. An ex vivo permeation protocol using porcine ear skin was implemented to assess the release of CBD and its penetration through the skin. Findings: Incorporation of the NEs in chitosan hydrogels does not significantly affect their structural properties as evidenced through SAXS, EPR and confocal microscopy. These findings indicate the successful development of a novel nanocarrier that preserves the NE structure with the CBD remaining encapsulated in the oil core while providing new rheological properties advantageous over NEs. Moreover, NE/HGs proved to be more efficient as a carrier for the release of CBD. Cell viability assessment revealed high biocompatibility of the proposed colloids