The School of Pharmacy Geneva-Lausanne (EPGL) – The First Ten Years

With the creation of the School of Pharmacy Geneva-Lausanne (EPGL) in 2003, cantons Geneva and Vaud pooled their resources with the objective of reinforcing the research and teaching in the pharmaceutical sciences. Its core research units cover all aspects of fundamental pharmaceutical research and include collaborative research with the University Hospitals of Geneva and Lausanne

Gender-Specific Differences between the Concentrations of Nonvolatile (R)/(S)-3-Methyl-3-Sulfanylhexan-1-Ol and (R)/(S)-3-Hydroxy-3-Methyl-Hexanoic Acid Odor Precursors in Axillary Secretions

The volatile fatty acid, (R)/(S)-3-hydroxy-3-methylhexanoic acid ((R)/(S)-HMHA), and the human specific volatile thiol, (R)/(S)-3-methyl-3-sulfanylhexan-1-ol ((R)/(S)-MSH), were recently identified as major components of human sweat malodor. Their 2 corresponding precursors were subsequently isolated from sterile and odorless axillary secretions. The purpose of this work was to analyze these 2 odor precursors in 49 male and female volunteers over a period of 3 years to elucidate to which extent they are implicated in the gender-specific character of body odor. Surprisingly, the ratio between the acid precursor 1, a glutamine conjugate, and the "sulfur” precursor 2, a cysteinylglycine-S-conjugate, was 3 times higher in men than in women with no correlation with either the sweat volume or the protein concentration. Indeed, women have the potential to liberate significantly more (R)/(S)-MSH, which has a tropical fruit- and onion-like odor than (R)/(S)-HMHA (possibly transformed into (E)/(Z)-3-methyl-2-hexenoic acid) that has a cheesy, rancid odor. Parallel to this work, sensory analysis on sweat incubated with isolated skin bacteria (Staphylococcus epidermidis Ax3, Corynebacterium jeikeium American Type Culture Collection 43217, or Staphylococcus haemolyticus Ax4) confirmed that intrinsic composition of sweat is important for the development of body odors and may be modulated by gender differences in bacterial compositions. Sweat samples having the highest sulfur intensity were also found to be the most intense and the most unpleasan

Evaluation of the immune response following a short oral vaccination schedule with hepatitis B antigen encapsulated into alginate-coated chitosan nanoparticles

The purpose of this work was to assess the ability of recombinant hepatitis B vaccine, encapsulated in alginate-coated chitosan nanoparticles, to induce local and systemic immune responses following oral vaccination. The antigen was administered either alone or in combination with the immunopotentiator, synthetic oligodeoxynucleotide containing immunostimulatory CpG motif (CpG ODN) as adjuvant, and associated or not with the alginate-coated chitosan nanoparticles. After two immunizations the group I (HBsAg associated with nanoparticles) and the group VI (HBsAg and CpG, both associated with nanoparticles) showed enhanced immune responses. Both groups showed significant higher values of the CD69 expression in CD4+ and CD8+ T-lymphocytes and lower values of this marker in B lymphocytes. Moreover, a strongest proliferative response of the splenocytes, ex vivo stimulated with concanavalin A, was observed in the same groups. Although with a presence of non-responder mice within the groups, only mice of the groups I and VI elicited the generation of anti-HBsAg antibodies detected in serum (IgG) and in the intestinal washings (sIgA). The results demonstrated that coated chitosan nanoparticles might have potential for being used as a deliver system for oral vaccination with the recombinant hepatitis B surface antigen.http://www.sciencedirect.com/science/article/B6T25-4PF1WCM-2/1/3fe2a6633c054a684fa0fafa7bb4a8b

"Pejzažni" prikaz nanolekova - ekspertska perspektiva

The field of nanotechnology is at the forefront of a scientific revolution, where the term “nano” transcends mere size and opens the door to enormous possibilities. In the context of drug development, the selection of a suitable drug delivery system (corresponding to a certain active pharmaceutical ingredient) is a pivotal decision. Accordingly, nanosystems have emerged as a promising avenue, offering innovative solutions, and gaining recognition for addressing healthcare issues. While these products hold immense promise, they have faced certain complexities in their translation from the preclinical to the clinical setting, reflected in the lack of proper assessment protocols for quality and safety aspects and, consequently, an insufficiently defined regulatory environment. Since the groundbreaking US Food and Drug Administration (FDA) approval of liposomal doxorubicin in 1995, approximately 80 nanomedicine products have received regulatory approval so far. Recent attention has gravitated toward lipid-based nanomedicines, particularly in the development of mRNA vaccines during the COVID-19 pandemic, further highlighting their significance. However, the relatively modest number of approved nanomedicines compared to the extensive research efforts raises important questions and underscores areas of uncertainty. This article provides an overview of the challenges in defining nanomedicines, their properties, the complexities of regulatory frameworks, and the imperative for standardized characterization protocols.Polje nanotehnologije se nalazi na čelu naučne revolucije, gde se termin "nano" izdiže iznad pukog označavanja veličine, otvarajući vrata novim mogućnostima. U kontekstu razvoja lekova, izbor odgovarajućeg sistema za isporuku / nosača (koji odgovara određenoj aktivnoj supstanci) predstavlja ključnu odluku. U tom kontekstu, nanosistemi već određeno vreme predstavljaju inovativna rešenja. Iako farmaceutski nanosistemi nose ogroman potencijal, suočavaju se sa određenim izazovima u pogledu translacije sa prekliničkog na klinički nivo, što se ogleda u nedostatku odgovarajućih protokola za ispitivanje kvaliteta i bezbednosti i, shodno tome, nedefinisanom regulatornom okruženju. Od revolucionarnog odobrenja liposomalnog doksorubicina od strane Američke agencije za hranu i lekove 1995. godine, pa sve do danas, oko 80 nano formulacija (nanolekova) odobreno je za kliničku primenu. Odnedavno je intenzivnija pažnja usmerena ka nanoformulacijama baziranim na lipidima, što je delom posledica razvoja mRNK vakcina tokom pandemije COVID-19. Međutim, relativno skroman nastup nanolekova na tržištu (u poređenju sa obimnim istraživačkim naporima i finansijskim ulaganjima u ovu oblast) otvara važna pitanja. Ovaj rad pruža pregled izazova u definisanju nanolekova, njihovih svojstava, kompleksnosti regulatornih okvira i imperativa za stvaranje standardizovanih protokola karakterizacije

Navigating towards improved cytotoxicity assessment in nanomedicine development: Shifting from colorimetric to fluorescence-based assays

It is well known that the characterization of nanomedicines can pursue different levels of complexity, both in the development stage and in the quality control process [1]. In line with physicochemical aspects, even more obstacles are encountered in biological safety assessment, while anticipation of their immunogenic potential represents an additional challenge. Moreover, interactions between the test reagents and the nanomaterial have been identified as one of the most important issues in toxicity testing that influence market authorization of nanomedicines, which ought to be resolved [1]. The European Nanomedicine Characterization Laboratory – the reference laboratory for nanomedicines, provides protocols for 2 colorimetric cytotoxicity assays employing LLC-PK1 (porcine kidney epithelial cells) and Hep-G2 (human hepatocarcinoma cells) cell lines. However, the latest recommendations in the field underline the demand for enhancing the testing procedures, while proposing incorporation of immune cells as target cell lines for toxicity evaluation, aiming to provide more reliable conclusions on nanomedicine safety in preclinical level. In this study 2 inherently different types of pharmaceutical nanosystemes were selected: nanoemulsion (NE) and solid lipid nanoparticles (LNP) and subjected to a set of orthogonal toxicity evaluation assays. Adjusted WST-1 (assessing mitochondrial activity as an indicator of cellular well-being) and LDH (lactate dehydrogenase release evaluation as an indicator of cell membrane damage) assays have been performed as the colorimetric tests, while propidium-iodide (PI)-based assay was developed as a fluore-scent counterpart (able to directly distinguish live from dead cells), using RAW 246.7 cell line (murine macrophages – immune system cell line). Starting concentration of the tested nanoformulations was 50 % v/v, and they were subsequently diluted with the factor of 2, to create a total of 8 concentrations. Incubation time was 4 h. Presented assays rely on completely different biological bases. Therefore, their careful combination can address some shortcomings in the in vitro evaluations established so far. Although similar toxicity trends were observed regardless the assay used, it was evident that the LDH assay required specific consideration. Since the supernatant is the subject of the analysis (not the cells directly), containing not only the enzyme of interest, but also the nanoformulations, in the wells corresponding to the 3 highest concentration of the NE/LNP pronounced scattering effects were observed. Such an event could be easily overlooked, potentially affecting the conclusions. However, it was overcome by careful design of control and blank wells (each test concentration was coupled with its own blank well containing no cells, but the same concentration of the NE/LNP in the culture medium). In contrast, absorbance measurements in WST-1 assay were performed in the absence of the NE/LNP, avoiding any interactions or scattering effects. Finally, developed PI-based assay proved to be the most relevant method. Based on the penetration of PI into the dead cell only, attaching to their DNA, the concentration of the dead vs. live cells could be directly estimated. What is more, after the incubation time, the measurements can be performed in the nanoformulation-free environment, surpas-sing the potential interactions. Notably, cell viability obtained in the PI-based assay followed the same trend as in the WST-1 assay but with significant difference in the obtained values for the first 3 concentrations (Figure 1).10th IAPC Meeting, Book of Abstract

Sizing experiments and bio-nano interactions: method matters

INTRODUCTION Physicochemical properties of many active ingredients jeopardize their pharmacological activity. To overcome identified obstacles, nanosystems as carriers for delivery of actives have been recognized as promising tools. Increasing number of applications for registration of nanotechnology-enabled pharmaceuticals and many more currently in preclinical or clinical studies raised some questions not only in the field of research and development, but also for regulators. Given the complexity of nanosystems, some specific challenges have been encountered in their characterization, which have not been fully addressed despite respectable research tradition in this field. Particle size and aggregation potential (especially in complex biological fluids) are some of the critical quality attributes of nanomedicines, being important in the context of physical stability of the colloidal system, and in terms of its safety profile and in vivo performance. Even though a bright future has been predicted for nanomedicines, some of the posted expectations have not been fully met so far. This might be reflected, at least at some points, in the certain methodological issues that commonly result in in vitro to in vivo translational gaps. This aspect underlines the importance of quality and safety assessment of nanomedicines which has also been recognized by globally leading research and regulatory bodies [1,2]. Therefore, the aim of the presented research was to perform a thorough analysis of the selected nanosystem (nanoemulsion) focusing on size estimation and particle-protein interaction applying several techniques, highlighting important factors for a reliable analysis. METHODLOGY Materials As a model nanosystem, previously developed nanoemulsion was used, containing medium-chain triglycerides (Mygliol 812, Fagron) as the oil phase, combination of polysorbate 80 (Acros Organics) and soybean lecithin (Lipoid S-75, Lipoid) as stabilizes, and highly purified water as the water phase. For protein interaction assessment, human serum albumin was used (HSA, Sigma Aldrich). Methods Nanoemulsion preparation Nanoemulsion was prepared via spontaneous emulsification, by dropwise addition of the mixture of the oil and stabilizers to the water phase under constant stirring. For nanoparticle-protein interaction assesment, nanoemulsion was incubated (1h, 37 °C) with HSA in the final concentration of 2.5 mg/ml. Sizing experiments – dynamic light scattering Size and size distribution (per se and in biorelevant environment) were evaluated applying batch mode dynamic light scattering (DLS, Zetasizer Nano ZS90, Malvern Instruments, UK), following the NCL guidance [3]. Intensity-based average hydrodynamic diameter (Z-ave) and polydispersity index (PDI) were analysed in line with relevant parameters of the method. Atomic force microscopy (AFM) Additional sizing analysis and morphological evaluation of the sample were performed applying AFM as a high-resolution technique. AFM analysis of the samples was performed by NTEGRA Prima atomic force microscope (NT-MDT, Moscow, Russia). Intermittent-contact AFM mode was applied using NT-MDT NSGO1 silicon, N-type, antimony doped cantilevers with Au reflective coating. Sample dilution corresponded to the optimal one selected for DLS, and 10 μl of the dilution was placed to the high-quality silica discs (Highest Grade V1 Mica Discs, Ted Pella Inc.) and dried in vacuum. Experiments were performed in the air, in contactless mode. Topographic images and “signal-error” images were collected, AFM images were created and analyzed with the software Image Analysis 2.2.0 (NT-MDT) and Gwyddion 2.60 (Free and Open Source software, Department of Nanometrology, Czech Metrology Institute). Small angle X-ray scattering (SAXS) SAXS experiments were performed with the general idea to analyze the structure of the dispersed nanodroplets more profoundly, and especially interactions in biorelevant surrounding (in contact with HSA). A laboratory X-ray setup was applied (Bruker Nanostar, Bruker AXS GmbH, Karlsruhe, Germany). Here, the Kα-line of a X-ray Cu source with a wavelength of 1.541 Å was used and further monochromated by a X-ray mirror. The beam was collimated to a beam diameter of approximately 0.4 mm using three pinholes. The sample-detector distance was set to 107 cm, which lead to a q-range of 0.07 ≤ q ≤ 2.3 nm-1. Calibration of the scattering vector q and estimation of the instrumental resolution of Δq = 0.25 nm-1 was done by measuring the first diffraction peak of a silver behenate sample. The scattered intensity was measured with an avalanche-based detector (VÅNTEC-2000, Bruker AXS). The transmitted part of the beam was determined using a home-made semi-transparent beam stop. The scattered intensity was extracted, radially averaged and integrated over all q-values using the Bruker software DIF-FRAC.EVA (Bruker AXS, version 4.1). The 1D data was transmission corrected and then background subtracted from the scattering of the solvent and the capillary using Matlab 2022. RESULTS AND DISCUSSION When applying DLS, as a preliminary technique, primary attention was put on the selection of optimal dilution level for the measurement, analyzing attenuation factor, count rate and intercept of the correlation function in different dilution ratios and with different dilution media (water, PBS 7.4 and 10 mM NaCl), and dilution 1:100 (v/v) was marked as the optimal one. However, significant differences in obtained nanodroplet size was observed depending on the type of medium. When water was used as a dilution medium, significantly higher Z-ave values were obtained (83.71±0.86 nm) compared to the situations where PBS 7.4 (73.50±0.75nm) or 10 mM NaCl (76.59±0.50nm) were used as dilution medium, indicating how sample preparation protocol might be crucial. Even though DLS was not sensitive enough to detect any interaction with HSA (no significant difference in terms of Z-ave and PDI compared to the results obtained in the same dilution medium without HSA), AFM captured qualitative difference in the droplet topography (Figure 1), raising ides on nanoemulsion interfacial interaction with HSA and increased aggregation potential. Further on, SAXS confirmed the existence of a bilayer structure as indicated by a prominent correlation peak at around 1 nm-1, which corresponds to a bilayer thickness of around 6.2 nm. SAXS (Figure 2; probably corresponding to the lecithin formations at the interface). It may be assumed that the bilayer structure changes its structure when mixed with HSA. CONCLUSION In this research, it has been demonstrated how important is to carefully select measurement conditions even for DLS -commonly used and the only standardized methods, in order to keep the measurements meaningful. Further on, not every method is capable of detecting some specific bio-nano interactions. Aiming to generate reliable datasets, condition sine qua non is to perform complementary techniques with increasing complexity. Further experimental segments should cover additional evaluation (e.g. analytical ultracentrifugation, thermal analysis, interfacial properties assessment, electron microscopy) that would shed light on bio-nano interactions important for in vivo fate of the nanosystems