@conference{
author = "Nikolić, Ines and Petrović, Marija and Krupnik, Leondard and Ranđelović, Danijela and Avaro, Jonathan and Neels, Antonia and Borchard, Gerrit and Jordan, Olivier and Đoković, Jelena and Savić, Snežana",
year = "2023",
abstract = "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.",
journal = "4th European Conference on Pharmaceutics, 20 - 21 March 2023, Marseille, France",
title = "Sizing experiments and bio-nano interactions: method matters",
url = "https://hdl.handle.net/21.15107/rcub_farfar_4571"
}
