Influence of surface characteristics on the in vitro stability and cell uptake of nanoliposomes for brain targeting

Influence of surface characteristics on the in vitro stability and cell uptake of nanoliposomes for brain targeting | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Influence of surface characteristics on the in vitro stability and cell uptake of nanoliposomes for brain targeting Dushko Shalabalija, Ljubica Mihailova, Nikola Geskovski, Andreas Zimmer, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4828653/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract In contemporary research, there is a clear emphasis on the physicochemical characteristics and effectiveness of nanoliposomal (NLs) formulations. However, there has been minimal focus on elucidating nano-bio interactions and understanding the behavior of these formulations at organ and cellular levels. Specifically, it is widely recognized that when exposed to biological fluids, nano-delivery systems, including NLs, rapidly interact with various biomolecules which have a significant impact on the functionality and destiny of the nano-systems but also influence cellular biological functions. Hence, the primary objective of this study was to illuminate the evolution of physicochemical characteristics and surface properties of NLs in biorelevant media. Additionally, in order to point out the influence of specific characteristics on the brain targeting potential of these formulations, we investigated NLs interactions with BBB (hCMEC/D3) and neuroblastoma cells (SH-SY5Y) under different conditions. The results obtained from in vitro comparative cell uptake studies on both cell culture lines after treatment with 3 different concentrations of fluorescently labelled NLs (5, 10 and 100 μg/mL) over a period of 1, 2 and 4 h showed a time- and concentration-dependent internalization pattern, with high impact of the surface characteristics of the different formulations. In addition, transport studies on hCMEC/D3/SH-SY5Y co-culture confirmed the successful transport of NLs across the BBB cells and their subsequent uptake by neurons (ranging from 25.17 to 27.54%). Fluorescence and confocal microscopy micrographs revealed that, once internalized, NLs were concentrated in the perinuclear cell regions. nanoliposomes surface characteristics stability cell uptake internalization cell co-culture Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Figure 10 Introduction Advancements in nanotechnology, coupled with the recognition of the benefits of utilizing nanoliposomes (NLs) as carriers for targeted delivery and controlled release of active components, suggest the use of these multifunctional platforms to address multiple pathologies associated with neurodegenerative diseases, such as Alzheimer's disease (AD), consequently impeding their progression [ 1 ]. Therefore, incorporating active components into these vesicles enhances their biological distribution, diminishes macrophage uptake in the reticuloendothelial system, and also lowers free drug concentrations, thus leading to a decrease in systemic toxicity. Adjustments to the composition and surface characteristics of NLs hold the potential for facilitating effective passage through the blood-brain barrier (BBB) as well as specific targeting of various brain structures [ 2 , 3 ]. However, despite the wide spectrum of advantages, only a limited number of NLs formulations designed for the treatment of brain diseases have successfully undergone the clinical evaluation and are currently available on the market [ 4 ]. This limitation could be attributed to a lack of comprehensive understanding of the crucial factors influencing the optimal delivery of active components to the central nervous system (CNS), particularly in the early stages of the development process. Furthermore, gaining a detailed insight and comprehension of the mechanisms governing the transport of NLs across the blood-brain barrier (BBB), along with the subsequent changes occurring during the delivery of active components to the brain, is imperative. Moreover, it is essential to discover how these kinetic processes are influenced not only by the properties of NLs formulations but also by the pathophysiology of the condition [ 3 , 5 ]. Taking into consideration the clinical potential of NLs as nano-carriers, European Medicines Agency (EMA) and U.S. Food and Drug Administration (FDA) issued guidances for the development of liposomal drug products, outlining several critical quality attributes (CQAs) that should be taken into consideration during the formulation and manufacturing stages. These CQAs encompass: identification and quantification of lipid composition; quantification of encapsulated, free, and total active component; characterization of NLs in terms of morphology, structure, particle size distribution, and surface charge; assess of the physical (fusion and aggregation) and chemical (lipid and active component degradation) stability and evaluation of the in vitro drug release kinetics [ 6 ]. Regarding the aforementioned part, in the contemporary research, it is evident that there is a significant emphasis on the physicochemical characteristics and efficiency of NLs formulations. However, minimal research has been dedicated to elucidating nano-bio interactions and comprehending the behavior of these formulations at organ and cellular levels. Namely, it is well known that, upon exposure to biological fluids, nano-delivery systems, including NLs, swiftly engage with various biomolecules, primarily involving three key aspects: (1) the adsorption of biomolecules onto the surface of nano-systems, leading to the formation of a protein corona (PC); (2) the reconstruction and alteration of functional proteins; and (3) redox reactions occurring between nano-systems and reactive species [ 7 ]. These interactions between nanomaterials and biological entities not only significantly impact the functionality and fate of the nano-systems but also influence cellular biological functions and may affect their targeting yields, the bio-distribution and cell internalization [ 8 ]. The current components of the serum could potentially exert significant deleterious effects on the lipid NLs structure, leading to the disruption of lipid bilayers and subsequent leakage of the encapsulated contents [ 9 ]. Conversely, research indicates that the adherence of plasma proteins to the surface of NLs may facilitate particle aggregation. As PC accumulates on the NLs' surface, it alters their surface characteristics. Consequently, the presence of proteins in the tissue milieu could potentially modify the cellular uptake mechanisms of both cationic and anionic nanocarriers. [ 10 ]. In simpler terms, our understanding of what precisely occurs to nano-delivery systems in a biological environment is limited. Questions linger regarding potential changes, such as alterations in their surface area, and how these modifications impact their interactions within the biological matrix. Therefore, of outmost importance is to assess the fundamental mechanisms governing reactions between the nano-carrier systems and the bioenvironmental components (nano-bio) in direction of devising strategies for manipulating these nano-bio reactions. In this context, monitoring the stability of nano-carriers in biological environments can be effectively achieved using advanced techniques such as asymmetric flow field-flow fractionation (AF4). AF4 has gained recognition as a crucial method for characterizing parameters such as particle size, polydispersity, drug loading, and overall stability of nanoparticle dispersions, covering a broad spectrum of particle sizes and sample polydispersity [ 11 ]. In recent years, collaborative efforts between the European Nanomedicine Characterization Laboratory (EUNCL) and the National Cancer Institute Nanotechnology Characterization Laboratory (NCI-NCL) have been pivotal. These collaborations have focused on developing robust Standard Operating Procedures (SOPs) tailored for measuring the physico-chemical properties of nanopharmaceuticals. This includes lipid-based nanocarriers such as liposomes and other (phospho)lipid nanoparticles, ensuring consistent and reliable characterization across different research settings and applications [ 12 ]. In addition to the aforementioned studies on the modified NLs surface, in vitro cell culture models offer a valuable avenue for gaining a deeper understanding of complex human organs like the brain and enhancing the translational relevance of in vivo models. Optimal selection of cell lines and fine-tuning experimental conditions serve as promising tools for comprehending the behavior of nano-delivery systems (NDS) upon contact with biological fluids and the microenvironment. Furthermore, these approaches facilitate the prediction of NDS' in vivo stability, toxicity, and therapeutic potential [ 13 ]. In this direction, hCMEC/D3 cell culture line is one of the in vitro models providing detailed insights into the uptake and transport of novel drug candidates and NDS across the BBB, influencing their subsequent therapeutic efficacy in the brain [ 14 ]. This model boasts advantages such as easy cell growth, mimicking basic BBB properties and morphological characteristics even without co-cultured glial cells [ 15 ]. Nonetheless, a major challenge lies in optimizing the tightness of tight junctions in cell monolayers to create a relevant in vitro model that mirrors human BBB characteristics, including permeability restriction with functional efflux and influx transporters and molecular exclusion [ 16 ]. Another relevant in vitro cell model used for predicting the in vivo behavior, internalization and performances of NDS in the brain tissues upon their successful transport across the BBB is the SH-SY5Y human neuroblastoma cell line which is characterized by rapid proliferation and non-expression of mature neuronal markers [ 17 ]. In this sense, this cell line can further be differentiated into neuronal like-morphologies by well-established protocols [ 18 ]. Similarly, as hCMEC/D3, undifferentiated SH-SY5Y cell culture line has been used as in vitro model for evaluation of the effect of neurotoxins as well as for investigation of NDS uptake and drug efficacy in the treatment of Alzheimer’s and Parkinson’s disease (Riegerová et al., 2021). This immature population of cells proliferate continuously as adherent catecholaminergic neuroblasts with a low portion of epithelial-like cells. It is important to mention that they do not possess the morphological, biochemical and functional properties of mature neurons [ 18 ]. It is important to emphasize that literature data on physical stability of NLs in different physiological barriers and compartments is limited. Therefore, the main aim of this research was to shed light on certain segments in the behavior of these carriers in a research that is designed in such a way to examine the evolution of the physico-chemical characteristics and their surface in biorelevant media. Additionally, the interaction of these carriers with relevant cell types under various conditions was examined to further evaluate whether the physico-chemical properties and stability of the prepared formulations affects their performance in an in vitro cell culture environment. For this purpose, detailed time- and concentration-dependent uptake experiments were carried out on hCMED/D3 and SH-SY5Y cell lines, separately, in order to evaluate the influence of the formulation properties as well as the extracellular microenvironment on the NLs internalization kinetics and mechanism in the brain tissues. A step forward was also made by conducting transport experiments on a co-culture system (hCMED/D3 / SH-SY5Y) with the aim to confirm the efficient transit of NLs across the BBB and their subsequent successful uptake into the neurons. Materials and Methods Materials Soybean lecithin (SL) was purchased from Vitalia (Skopje, N. Macedonia) and LIPOID PE 18:0/18:0-PEG 2000 (PEG) from Lipoid (Ludwigshafen am Rhein, Germany). Cholesterol (CH), Dulbecco’s Phosphate Buffered Saline (DPBS), collagen type I from rat tail, fluorescein isothiocyanate isomer I (FITC) and Nile Red dye were obtained from Sigma Aldrich (St. Louis, USA). Immortalized human cerebral microvascular endothelial (hCMEC/D3) cell line (CELLutions, Biosistems/Cedarlane®, Canada) were maintained in Endothelial Basal Medium-2 (EBM-2), supplemented with Fetal Bovine Serum (FBS), chemically defined lipid concentrate, HEPES 1M and penicillin − streptomycin (Life Technologies, California, USA), human basic Fibroblast Growth Factor (bFGF), ascorbic acid and hydrocortisone (Sigma Aldrich, St. Louis, USA) and detached with trypsin-EDTA (GIBCO, Thermo Fisher Scientific, California, USA). Human neuroblastoma cell line (SH-SY5Y) was purchased from LCG Standards (Wesel, Germany) and maintained in Dulbecco's Modified Eagle Medium (DMEM) (Thermo Fisher Scientific, California, USA). EndoGRO-MV SCME004 complete media kit was provided by Merck (Merck Group, Germany). CellTiter 96 AQueous Non-Radioactive Cell Proliferation Assay (MTS) and CytoTox-ONETM Homogeneous Membrane Integrity Assay (CytoTox) were obtained by Promega (Wisconsin, USA). Alexa FluorTM Phalloidin 488, Hoechst Fluorescent stain and Dil stain (1,1'-Dioctadecyl-3,3,3',3'-Tetramethylindocarbocyanine Perchlorate ('DiI'; DiIC18(3))) were purchased from Thermo Fisher Scientific (California, USA). All chemicals and reagents used were of the highest purity grade commercially available. Preparation of the nanoliposomes (NLs) NLs samples (SL:CH:PEG = 8.71:1:0; 8.71:1:1.67 and 8.71:1:0.67 molar ratios for NLb0, NLb1 and NLb2, respectively) were prepared by modified lipid film hydration technique as described by Shalabalija et al. (2021). All fluorescent dyes, Nile Red (1.6%, w/w ) and DIl stain (2.5%, w/w ), used for the purpose of the cell culture experiments were added in the organic phase during the preparation process according to Mihailova et al. (2023). Particle size, particle size distribution and z-potential of the NLs Тhe z- average diameter, the polydispersity index (PDI) and z-potential (ZP) were determined using a Zetasizer Nano Series, Nano-ZS, (Malvern Instruments Ltd., UK), after diluting the optimal NLs samples in 10 mM PB pH 7.4 (1:20, v/v ). Measurements were made under the following conditions: 25°C, thermostating time of 120 seconds, viscosity of the medium 0.8894 cP, dielectric constant 78.5 and an angle of 173°. At least 3 separate preparations (batches) from each sample were measured in triplicate. Results of each analysis was the average of 12 consecutive measurements. Stability studies of NLs in cell culture medium The samples (NLb0, NLb1 and NLb2) at a native concentration of 30 mg/ml were firstly diluted to a final concentration of 1 mg/ml (total volume of 2 ml) in cell culture medium (Endo-GRO-MV, Cat SCME004, Sigma Aldrich), and then incubated at 37°C for 1 and 4 h in an Eppendorf thermomixer under constant stirring (300 rpm). Incubation took place in serum free and 5% serum supplemented cell culture medium (prepared according to the manufacturer's instructions). At the end of the incubation time, the samples were vortexed (5 seconds) and a volume of 1 ml was transferred to a vial for asymmetric flow field-flow fractionation analysis (AF4 analysis). Experiments were carried out by in-line coupling the AF4 system with UV/VIS, MALS and DLS detectors. The AF4 system used was composed of an Eclipse Dualtec separation system (Wyatt Technology Europe GmbH, Dernbach, Germany) and an Agilent 1260 Infinity high-performance liquid chromatograph (Agilent Technologies, Santa Clara, USA) equipped with a degasser (G1322A), an isocratic pump (G1310B), autosampler (G1329B) and multiple wavelength (MWD) detector (G1365C) ​​set at 230 nm. In the Eclipse SC separation channel, regenerated cellulose membranes (10 kDa) and a spacer height of 350 µm were used. Phosphate buffer (PBS) was used as eluent. The detector flow rate was set at 0.5 ml/min and the injection volume was 50 µl. Separation settings were: a) Elution 0–3 mins; b) Focus: 3–5 mins (Focus flow: 1.0 ml/min); c) Focus + Inject: 5–10 mins (Focus flow: 1.0 ml/min); d) Elution 10–50 mins (Cross-flow from 1.0–0.1 ml/min); e) Elution 50–55 mins (Cross-flow 0.1 ml/min); f) Elution 55–60 mins (Cross-flow 0.1–0.0 ml/min); g) Elution: 60–70 mins (Cross flow 0.0 ml/min). The outlet of the MWD detector was connected to a DAWN 8 + HELEOS II multi-angle light scattering detector (MALS) operating with a 658 nm laser (Wyatt Technology Europe). A DLS (Malvern Zetasizer Nano-S, UK) with an installed quartz flow-cell (ZEN0023) was also used in this study in flow mode. Refractive index (RI) and absorption parameters were set to 1.38 and 0.010 respectively. Water was set as the dispersant, the temperature was set to 25°C, while the attenuation was set to 11. 'General purpose (normal distribution)' was chosen as the analysis model. Proteomic profiling of the adsorbed proteins onto NLs surface Protein electrophoresis was performed by 2100 Bioanalyzer using High Sensitivity Protein 250 kit assays in reducing condition according to the manufacturer’s instruction. Briefly, samples prepared as for the AF4 analysis, at the end of the incubation time, were equilibrated using the standard labelling buffer and labelled fluorescent dye. Next, samples were diluted 200-fold in milliQ-water and denatured at 95°C for 5 min in reducing condition by adding 3.5 µl of 1 M Dithiothreitol (DTT) buffer solution to 100 µl of each sample buffer. After cooling, samples were loaded on the microfluidic chip for electrophoresis, in accordance with the manufacturer's instructions. All reagents and instruments were from Agilent Technology, California, USA. hCMEC/D3 and SH-SY5Y cell culture lines Both cell culture lines were seeded and cultivated according to the Supplier’s guidelines, explained in details in Mihailova et al. (2023). In vitro uptake and internalization experiments were conducted using hCMEC/D3 cell cultures. T-75 cell culture flasks from Greiner Bio-One GmbH, Germany, were initially coated with 0.05 mg/ml rat tail collagen type I in DPBS and left to stand for at least 1 hour at 37°C. The cells were cultured in supplemented EBM-2 at 37°C with 5% CO 2 . Medium replacement occurred every 2–3 days until cells reached confluence. Upon reaching confluence, the medium was aspirated, and cells were detached from the flask walls by incubating them at 37°C for 8 minutes in 0.1 mg/ml trypsin-EDTA solution. The cell suspension was then centrifuged at 1500 rpm for 3 minutes, and the supernatant was discarded. The cells were resuspended in 5 ml of cell medium and were prepared for further experimentation. The human neuroblastoma cell line was cultured in DMEM at 37°C in a 5% CO 2 atmosphere. Medium replacement occurred every 2–3 days until cells reached confluence. During the splitting process, the medium was aspirated, and cells were detached from the flask walls after incubating at 37°C for 3 minutes in 0.1 mg/ml trypsin-EDTA solution. The cell suspension was then centrifuged at 800 rpm for 5 minutes, and the supernatant was discarded. The cells were subsequently resuspended in 5 ml of cell medium, making them ready for further experiments. Cell uptake assessment of NLs In order to determine the influence of the PEG amount and exposure times on the uptake of the NLs, hCMEC/D3 or SH-SY5Y cells were seeded in 96-well plates at a density of 10 4 cells/well in their respective cell culture medium (200 µl/well) and incubated for 48 h at 37°C and in the presence of 5% CO 2 . As previously described, wells for hCMEC/D3 cells were pre-coated with collagen type 1 (0.05 mg/ml) for 1 h. After reaching confluence, the cell culture medium was replaced and the cells were treated with different concentrations of the fluorescent dye labeled formulations NLb0, NLb1 and NLb2 with Nile Red (Sigma Aldrich, USA) (previously diluted with PBS and dispersed in the appropriate cell culture medium at final concentrations of 5, 10 or 100 µg/ml). Incubation was performed for 1, 2, and 4 h (37°C, 5% CO 2 ), followed by washing step with PBS and lysis with 2% Triton X-100 (2 h, 37°C, 5% CO 2 ). The resulting fluorescence was measured on a plate reader at 535 nm excitation and 635 nm emission (BMG Labtech, Ortenberg, Germany). Untreated cells were used as blank. The quantitative amount of internalized NLs was calculated according to pre-obtained regression analysis equations constructed from the fluorescence obtained over a range of concentrations for each of the formulations (non-incubated with cells). The experiments were performed three times on at least six replicates from each sample. Cell uptake assessment of NLs in presence of transport pathways inhibitors In order to determine the exact mechanism of cellular internalization, uptake experiments were also performed in the presence of inhibitors for specific transport pathways. Cells were seeded and cultured as described above. The quantitative amount of the internalized NLs was investigated on both cell lines. One set of cells was treated with 15 µM chlorpromazine and another set with 25 µM indomethacin for 40 min, followed by incubation with NLs (at final concentrations of 5, 10 or 100 µg/ml) for 2 h (37°C, 5% CO 2 ). Third set of cells was left at 4°C for 40 min, before NLs incubation in cold condition for 2 h. The resulting fluorescence was measured as described previously (same conditions as for the 37°C experiments), and the quantitative amount of internalized NLs was calculated and expressed as the % of the uptake of the corresponding NLs concentrations at 37°C (taken as 100%). The experiments were performed three times on six replicates from each sample. 1.1. Cell uptake experiments on co-cultured hCMEC/D3 and SH-SY5Y cell line The hCMEC/D3 cells were seeded on Transwell inserts (5x10 4 cells/insert), previously coated with 0.05 mg/ml type I collagen in DPBS (1 h, 37 o C) and incubated with EBM-2 (0.5 ml cell medium in the apical part of the insert and 1 ml in the basal part of the well). The medium was changed every 2–3 days until a transendothelial resistance (TEER) value > 230 Ω was reached, indicative of solid monolayer formation and tight junctions between endothelial cells [ 16 ]. In parallel, SH-SY5Y cells were seeded in 12-well plates (3x10 4 cells/well) and cultured in 1 ml DMEM for the same period of time, until reaching confluence. Furthermore, the inserts with hCMEC/D3 cells were placed in the plates with formed monolayers of SH-SY5Y. NLs previously diluted in PBS and dispersed in EBM-2 (final concentration 10 µg/ml) were added to the apical part of the inserts, and 1 ml of DMEM was added to the basal part of the plates. After 2 h of incubation (TEER > 210 Ω), the inserts were removed and the SH-SY5Y wells were washed twice with PBS and lysed with 2% Triton X-100 (2 h, 37°C, 5% CO 2 ). The resulting fluorescence was measured on a plate reader at 535 nm excitation and 635 nm emission (BMG Labtech, Ortenberg, Germany). The wells with SH-SY5Y incubated in the corresponding cell medium (not treated with NLs) were used as blanks. The quantitative amount of NLs taken up was calculated as % of the fluorescence of the native nano-formulations (in the same concentrations). The experiments were performed three times on at least six replicates from each sample. Internalization studies a) Internalization studies of NLs in live hCMEC/D3 and SH-SY5Y cell lines by fluorescence microscopy In order to obtain a deeper insight of the internalization and co-localization of the prepared NLs samples in living cells, hCMEC/D3 and SH-SY5Y were first seeded on 35-mm glass dishes at a density of 2x10 5 cells per well (µ-Dish 35 mm, WillCo Glass Bottom Dishes, Netherlands) and subsequent incubation for 48 h (37 o C and 5% CO 2 ) was conducted. Afterwards, the medium was removed, and the cells were treated with the Nile Red pre-labeled NLs (NLb0, NLb1, and NLb2) dispersed in the respective cell culture medium at a final concentration of 10 µg/ml. After 1, 2 and 4 h incubation (37°C, 5% CO 2 ), the cells were washed twice with PBS, followed by subsequent fluorescence microscopy analysis at 37°C (Zeiss Axio Observer Z1 inverted microscope, Zeiss, Jena, Germany), equipped with an epifluorescence illuminator and a plate heating chamber. The resulting images were processed using Carl Zeiss software (ZEN 2.6). In this direction, another set of experiments was conducted, where SH-SY5Y cells before the incubation with pre-labeled NLs with Dil (1,1'-Dioctadecyl-3,3,3',3'-Tetramethylindocarbocyanine Perchlorate) (10 µg/ml, 4 h, 37°C, 5% CO 2 ), were incubated with pHrodo Green dextran conjugate characterized by green fluorescence in an acidic environment (for visualization of endocytic pathways). Further, the cells were stained and visualized as described above. b) Internalization studies of NLs in lhCMEC/D3 cell line by confocal microscopy Confocal laser scanning microscopy (Carl Zeiss, Axiovert 200M Inverted Microscope) was performed in order to confirm the internalization of NLs inside the cells and their co-localization in the cellular structures of BBB. For this purpose, cells were seeded on 35-mm glass dishes (µ-Dish 35 mm, WillCo Glass Bottom Dishes, Netherlands) at a density of 2x10 5 cells per well and incubated for 24 h (37 o C and 5% CO 2 ). After incubation (4 h, 37° C and 5% CO 2 ) with the previously labeled NLs with Dil fluorescent dye, the cells were washed twice with PBS and fixed with 3.7% paraformaldehyde for 20 minutes at room temperature. After washing the cells with PBS, cytoskeleton was stained with Alexa FluorTM Phalloidin 488 green dye (6.6 µM) in 1% bovine serum albumin (10 minutes, 37 o C), followed by nuclear staining Hoechst (hCMEC/D3), for 5 minutes at room temperature. Before microscopic visualization, Vectashield mounting medium was added to maintain and preserve the fluorescent dye. Images were processed using Carl Zeiss software (ZEN 2.6). Statistical analysis Statistical analysis of the obtained results for quantitative uptake of NLs was carried out by implementing the method of least squares (PLS) using validated statistical software Simca 14.1 (Sartorius Stedim Biotech, Germany). In order to highlight the dominant independent variables that have a significant effect in the model, the VIP score was used. Results and Discussion Particle size, particle size distribution and z-potential of the NLs Several studies have reported that despite the composition and the other surface properties (i.e. surface charge), the size range of long time circulating NPs may strongly affect their stability, in vivo circulation time, as well as BBB retention and the possibility of entering into specific interactions with the BBB structures, thus their transport and uptake by the brain cells [ 21 ]. In this direction, one of the initial segments was determination of the z-average diameter and z-potential of NLs in order to further correlate them to the outcome of cell uptake experiments. The z-average (hydrodynamic) diameter of the prepared NLs formulations with different amounts of PEG coated on the surface was in a range from 115–130 nm with PDI < 0.3 (Table 1 ) which indicates a narrow unimodal size distribution of the NLs in all formulations. According to the literature data, liposomal nano-vesicles from 100 to 140 nm exhibit longer half-life in blood circulation and avoidance of PC formation, when compared to nano-vesicles with diameter > 200 nm, and also, better encapsulation efficiency than liposomes < 100 nm [ 22 ]. On the other hand, in the study of Nowak et al. (2019) was confirmed that spherical particles around 120 nm associate with the endothelium approximately 30-fold more than 200 nm particles, which is of extreme importance for their successful transport across BBB and consequently, efficient treatment of CNS diseases [ 23 ]. Table 1 Physical characterization of prepared NLs (n = 6) Formulation z-average [d h , nm] PDI Zeta potential [mV] NLb0 127.25 ± 0.12 0.277 ± 0.00 -51.96 ± 2.22 NLb1 114.90 ± 0.92 0.238 ± 0.00 -15.77 ± 0.51 NLb2 131.03 ± 0.30 0.289 ± 0.04 -37.64 ± 1.42 Table 1 From Table 1 , it can be also observed a decreasing trend of the negative z-potential with the increase of PEG amount on the surface of NLs. As previously reported in our earlier study, this reverse trend is probably result of the reduced NLs electrophoretic mobility due to the hydrodynamic resistance given by PEG presence which also contributes for masking the predominant negative charge of the structural phospholipids present in the NLs (Shalabalija et al., 2021). Stability studies of NLs in cell culture medium In order to provide a more detailed examination of the influence of serum components present in the cell culture medium on the average NLs size, AF4-MALS/DLS analysis was performed. For this purpose, the particle size of the native formulations (NLb0, NLb1 and NLb2) was first determined, as well as after their incubation with serum-free and serum supplemented cell culture medium over 1 and 4 h. Prior performing these fractionation analyses, the z-average mean diameter of the NLs was also examined by dynamic light scattering in batch-mode, and ranged from 96.10 ± 0.81 to 140.20 ± 0.95 nm (PDI < 0.256). Literature data suggests that Dynamic Light Scattering (DLS) is widely employed for sizing liposomes and other colloidal materials. It operates by detecting laser light scattered due to Brownian motion of particles or macromolecules in suspension, with the scattering frequency dependent on particle size, offering rapid and straightforward analyses. However, larger particles can skew results and complicate measurements in heterogeneous samples, typically requiring a five-fold difference in average size to resolve distinct populations. In contrast, Asymmetric Flow Field-Flow Fractionation (AF4) is more time-intensive but excels in fractionating samples under optimal flow and separation conditions. This method is distinguished by gentle separation conditions and a wide operational range. Unlike conventional chromatography, AF4 does not utilize a stationary phase. The separation mechanism involves a longitudinal parabolic flow profile whithin the channel.This profile induces smaller particles to elute more swiftly compared to larger particles, particularly in proximity to the semipermeable membrane [ 24 ]. In particular, AF4 is a precise method for separating liposomes based on their hydrodynamic size, with particle sizes determined directly from their elution times. AF4-MALS has been extensively utilized for sizing various categories of nanoparticles such as metal oxides, polymeric and silica nanoparticles. Additionally, it has been employed for the separation of diverse macromolecules and structures including proteins, viruses and cells. These applications have facilitated the analysis of liposomes, enabling the separation of populations obtained from the same method synthesis and determination of their size. Optimizing separation variables in AF4-MALS involves several parameters such as cross-flow conditions, focusing rate and duration, sample loading, and carrier conditions. The composition of the carrier buffer, as well as its ionic strength and pH are crucial considerations for stabilizing the structures, preventing agglomeration or sedimentation, and avoiding interference with analytes and the membrane [ 11 ]. Figure 1 represents the fractograms obtained from the AF4-analysis of the native formulations. The black line originates from the UV signal (230 nm) and the red line from the light scattering signal at 90°. Both signals are normalized to the highest signal. The UV-absorbance peaks (black signal), observed at a retention time of about 20 minutes, are most likely related to the absorption of the PEG. In addition, it can be seen that a more intense absorption peak is obtained at the same retention time for NLb1, the formulation with the highest amount of PEG on the surface, in comparison to NLb2. Moreover, it can be observed that the shape of the light-scattering (red line) and UV-signals (RT 40–60 min) of those materials treated with little or no PEG (NLb0 and NLb2) looks differently compared to NLb1 (pyramid-like vs. near Gaussian like). As already mentioned in several occasions, the presence of PEG on the surface of NLs can improve the physical stability of the liposomal dispersions through steric repulsion [ 25 ]. Therefore, formulations with a low amount of PEG or no PEG tend to agglomerate or lose their native structure thus leading to fragmentation. Hence, the unusual peak-shape at 20 min and the weak signal at around 60 minutes in NLb0 may be a result of the absorption of various fragments of the nanoliposomes and/or some of the components present in the soybean lecithin, as well as artefact of the initial formation of peroxides in the unsaturated fatty acid residues of the phospholipid molecules which show maximum absorbance at around 230 nm [ 26 ]. Figure 1 In Figure S1 , the light scattering signal at 90° and the related geometrical radii are overlaid for native formulations NLb0, NLb1 and NLb2. Formulation NLb1 (red lines) showed to contain slightly larger particles in the final part of the eluted peak compared to the other two formulations, probably attributable to some small, insignificant fraction of agglomerated NLs formed during the measurements. The same was confirmed by DLS in-line measurements (Fig. S2). Each of the four sections (a-d) included in Fig. 2 show overlaid signals obtained from the UV-detector and the z-average coming from the DLS operated in flow-mode. It can be observed that there is no significant change in the size of NLb1 vesicles (formulation with 50 mg PEG), after their incubation in cell culture medium without and with serum for a period of 1 and 4 h. This situation only confirms the stability of this formulation, and also supports the fact that PEG contributes in the prevention and suppression of the PC formation process. Figure 2 During these experiments, it was also observed that formulation NLb2 incubated for 1h in the serum supplemented cell culture medium contained slightly smaller particles in the upper particle size range compared to the same sample incubated for 4 h (Fig. 3 b). In addition, the measurements also showed smaller NLb2 particles in the upper size range when this formulation was incubated in serum supplemented cell medium compared to when it was incubated in serum free cell medium at the both time points, separately (1 and 4 h) (Fig. 3 a, c). In the case of particle diameter increase as a result of the PC formation, it would be expected a general increase in size throughout the whole size range to be seen. In this case, the increase was only observed in the upper size range. In this direction, the obtained results can be attributed to the fact that serum proteins can also stabilize nano-carriers, thus preventing their aggregation process (Kennedy et al., 2018). Figure 3 Similar to NLb2, AF4 analysis showed that the particles of NLb0 (non-PEGylated formulation) were characterized by a smaller size when incubated in serum supplemented cell culture medium, compared to those incubated in serum free medium. As already discussed, this situation can be the result of the stabilizing effect provided by the proteins present in the serum on the NLs (Fig. 4 a, c). However, it is interesting to note that during these studies an unexpected decrease in the size of NLs was observed after 4 h vs. 1 h of incubation in serum supplemented medium (Fig. 4 b), which is probably due to the fact that PC formation is a dynamic process that generally tends to evolve over time and involves many different driving forces controlled by the properties of nano-systems, proteins and the medium itself [ 27 ]. The obtained results are in accordance to the results of the study of Miclăuş et al. (2014), where it was demonstrated that the soft corona (formed at the initial time points of incubation) contains more proteins than the hard corona formed at later time intervals, resulting in a larger particle diameter at early incubation periods [ 28 ]. Figure 4 Proteomic profiling of the adsorbed proteins onto NLs surface In the next step, qualitative analysis of the adsorbed serum components on the surface of the nano-formulations (NLb0, NLb1 and NLb2) was investigated (Fig. 5 ). For this purpose, NLs were incubated in cell culture medium with and without serum (as control) for 1 and 4 h. From the graphical representations, it can be observed that the protein adsorption by NLb1 and NLb2 (Fig. 5 ) is already expressed in the first hour of incubation, resulting with strong bands at about 60 kDA, originating from albumin, the most abundant protein in the serum. On the other hand, these bands are not so expressed in NLb0. Considering that the sensitivity of the bioanalyzer is high and it covers a wide range of concentrations, the results obtained for this formulation may be due to problems with denaturation of proteins present in the formed PC, as well as the manipulation and processing of the sample. Namely, false negatives might arise because proteins detach from the nanoparticle-corona complex under the influence of centrifugal forces. Hence, it's crucial to ascertain the optimal number of washing cycles and centrifugation duration necessary for effectively isolating a particular type of nanosystem-corona complex from a protein-rich medium [ 29 ]. In addition, weak bands from other proteins can be observed in all three formulations, but more detailed analysis by mass spectrometry is required. Figure 5 Cell uptake assessment of NLs As previously discussed, one of the prerequisites for achieving a therapeutic effect in the brain is the successful transport of NLs across the BBB, as well as their internalization in neurons. In this direction, after determining the safety concentration range of NLS [ 30 ], we then investigated the in vitro cell uptake of NLs by two cell lines: BBB cells (hCMEC/D3) and human neuroblastoma cells (SH-SY5Y). Quantitative uptake experiments performed on the two cells lines (hCMEC/D3 and SH-SY5Y) exposed to 5, 10 or 100 µg/ml of the NLs under investigation and at different time-points (1, 2 and 4 h) are reported in Table 2 . As it can be observed, there is a gradual increment of the cell uptake for all formulations with the increase of their concentrations at all-time points for both cell lines analyzed, which is an expected phenomenon. Similar, increasing trend in the uptake can be seen with prolonged incubation time, except for the highest concentration tested (100 µg/ml), for which differences were found between the two cell lines. At this concentration, the uptake measured of all NLs formulations was around 3 µg at all incubation times for hCMEC/D3 cell line, whereas for SH-SY5Y it varied from 3.28 ± 0.21 to 4.14 ± 0.19 µg at 1 and 4 h, respectively. It is well known that the internalization of nanoliposomes into cells can take place through several energy dependent endocytic pathways (phagocytosis, clathrin-mediated endocytosis, caveolin-mediated endocytosis, clathrin/caveolae-independent endocytosis, and micropinocytosis), as well as passive transport or diffusion which is an uncompetitive movement of the nano-systems, either directly through membrane phospholipids (simple diffusion) or in combination with membrane proteins (facilitated diffusion) [ 10 , 31 ]. Numerous studies have revealed that endocytosis is a process that occurs through membrane-particle adhesion followed by elastic deformation of the cell membrane and receptor diffusion to the surface of the membrane, processes highly dependent on the physico-chemical properties of the NLs, as well as their concentration and exposure time (Sabourian et al., 2020). As no significant increase of uptake was found for the highest concentration tested for all three NLs and for both cell lines, it is expected that at 1 h, hCMEC/D3 and SH-SY5Y cells have already reached their maximal endocytic potential, which is probably due to the saturation of uptake mechanisms leading to limited internalization [ 32 – 34 ]. Additionally, as previously elaborated, the increased uptake by increasing the incubation time observed at 5 and 10 µg/ml for all NLs investigated confirms the lack of a saturable transport process [ 35 ]. Table 2 Cell uptake of NLs (µg) by hCMEC/D3 and SH-SY5Y cell lines hCMEC/D3 cell line SH-SY5Y cell line 1h 2h 4h 1h 2h 4h 5 µg/ml NLb0 0.09 ± 0.03 0.17 ± 0.01 0.54 ± 0.09 0.19 ± 0.03 0.28 ± 0.01 0.31 ± 0.02 NLb1 0.22 ± 0.03 0.38 ± 0.03 0.55 ± 0.05 0.18 ± 0.02 0.24 ± 0.01 0.24 ± 0.01 NLb2 0.20 ± 0.02 0.23 ± 0.05 0.29 ± 0.04 0.18 ± 0.02 0.20 ± 0.01 0.21 ± 0.01 10 µg/ml NLb0 0.36 ± 0.01 0.59 ± 0.04 1.07 ± 0.12 0.61 ± 0.11 0.74 ± 0.04 0.85 ± 0.04 NLb1 0.46 ± 0.05 0.64 ± 0.01 1.12 ± 0.02 0.61 ± 0.06 0.66 ± 0.03 0.74 ± 0.02 NLb2 0.51 ± 0.02 0.56 ± 0.01 0.70 ± 0.12 0.51 ± 0.01 0.66 ± 0.08 0.72 ± 0.04 100 µg/ml NLb0 3.20 ± 0.13 3.38 ± 0.03 3.25 ± 0.06 3.67 ± 0.10 3.85 ± 0.10 4.14 ± 0.18 NLb1 2.92 ± 0.01 3.02 ± 0.07 3.06 ± 0.03 3.28 ± 0.21 3.27 ± 0.12 3.59 ± 0.07 NLb2 2.98 ± 0.07 3.03 ± 0.02 3.08 ± 0.05 3.57 ± 0.25 3.58 ± 0.10 3.81 ± 0.11 Table 2 In order to investigate the influence of the possible independent factors (experimental conditions – time of incubation/NLs concentration, type of formulation-amount of PEG) on the quantitative uptake of the NLs in the specified cell line cultures, a multivariate statistical analysis was performed. The correlation coefficients obtained from the initial model, comprised of all internalization data (normalized uptake - %) were low, but it was observed that the cell type predominantly affects the scores of the individual points, which is why it was decided PLS-DA to be performed. In continuation to the above discussed, for the internalization kinetic experiments, separate multivariate statistical models were performed for each cell culture. The internalization model in hCMEC/D3 also confirmed that the sample concentration and the exposure time were dominant factors on the percentage of NLs taken up (Fig. S3 a-c). According to the VIP plot the formulation type i.e. the amount of PEG on the surface also had a significant effect on the uptake (S3 d). The model of kinetic experiments on the SH-SY5Y cell line presents a similar behavior to the previous model, where concentration and exposure time were the dominant factors affecting uptake, while the amount of PEG on the surface has a smaller but distinctive influence (Fig S4 a-d). As it can be seen from Table 2 , the uptake of NLs by hCMEC/D3 after 4 h of incubation is highest for the formulation with the highest amount of PEG on the surface - NLb1 (0.55 ± 0.05 µg and 1.12 ± 0.02 µg, at 5 and 10 µg/ml treatment concentrations, respectively), followed by the formulation with no PEG on the surface - NLb0 (0.54 ± 0.09 and 1.07 ± 0.12 µg, at 5 and 10 µg/ml treatment concentrations, respectively). The lowest rate of internalization was observed in the formulation with 5 mg of PEG on the surface - NLb2 (0.29 ± 0.04 and 0.70 ± 0.12 µg, at 5 and 10 µg/ml treatment concentrations, respectively). Numerous research groups have demonstrated that the PC formation can significantly influence and dictate the cell recognition and the internalization, as well as the intracellular trafficking of nano-systems since it gives them new biological identity [ 36 ]. Adsorbed proteins on nanostructures can hinder cell membrane adhesion and compromise stability, leading to decreased cellular uptake. The composition of formed PC on NLs affects targeting yields, nanoparticle-cell interactions, and internalization mechanisms. Nano-carriers can induce structural changes in adsorbed proteins, influencing cell signal transduction. The impact of PC on particle-cell interactions varies based on particle properties and cellular components, as well as the nature of the cell culture medium [ 37 – 39 ]. The medium used for the cell uptake experiments of hCMEC/D3 cells was supplemented with serum as well as growth factors, dyes and antibiotics. Apart this fact, the highest cell uptake of NLb1 can be also attributed to its z-potential which is less negative (-15 mV), compared to the other two formulations, since less negative or positively charged nano-carriers would be expected to be more efficient in crossing BBB which is characterized by a negative charge [ 40 ]. On the other side, several research groups have shown that the PEG surface density and conformation play a key role and improve the diffusion and transport of different types of nano-systems across endothelial barriers, particularly the BBB, and consequently, their brain distribution. Taking into consideration that NLb1 exhibits high amount of PEG, the density of the chains on the liposome surface is expected to be increased and be characterized with “dense brush” conformation as steric hindrances restrict movement and self-coiling of the grafted polymer [ 41 ]. In the research of Nance et al. (2012) it was also demonstrated that nano-systems characterized by “dense brush” PEG coating can permeate the BBB and accumulate more efficiently in the brain parenchyma ex vivo, than the uncoated [ 42 ]. From Table 2 it can also be observed that during 1 h incubation intervals the cell uptake amount of NLb0 is lower compared to NLb2, at both treating concentrations (5 and 10 µg/ml). However, the opposite case is noticed over 4 h incubation where the quantitative cellular uptake of NLb0 was approximately 1.5 fold higher than the uncoated formulation. The main reason for this can be the fact that in our previous stability studies conducted by AF-4 analysis, it was shown that serum proteins present in the cell culture medium stabilize NLb0 in terms of preventing the process of aggregation, and additionally, unlike for NLb2, there was a decrease in the average diameter over the incubation time of 4h, which is probably due to the dynamic process of PC formation. These results only confirm the statement regarding the opposite dependence between the particle size and hCMEC/D3 liposomal uptake and adhesion as well as the alterations on the internalization promoted by the adsorbed serum proteins onto NLs surface [ 43 ]. On the other hand, NLb2, despite the low amount of PEG on its surface also showed saturable uptake within the first hour (0.20 ± 0.02 and 0.51 ± 0.02 µg, at 5 and 10 µg/ml treating concentrations, respectively), since the difference in the amount of internalized particles of NLb2 in the later incubation times (0.29 ± 0.04 and 0.70 ± 0.12 µg after 4 h, at 5 and 10 µg/ml treating concentrations, respectively) tended to fade out and become significantly lower compared to the other two formulations. These results additionally confirm the limited capacity of NLb2 to accumulate intracellularly and is also indicative of equilibrium between endocytosis and exocytosis [ 44 ]. Additionally, literature data suggests that PEG-coated particles with a surface charge between − 20 and − 40 mV are not capable to cross BBB probably due to the insufficient dense coating of PEG (Nance et al., 2012). This is in accordance with our results since we can conclude that hydrophilicity as well as surface charge can significantly affect the nano-system delivery to BBB, and thus brain tissues. When it comes to the cellular uptake of the NLs by SH-SY5Y cell line, there is a different trend of quantitative internalization among the formulations. Namely, the formulation characterized by the highest cellular uptake after 4 h is the non-PEGylated NLb0 (0.31 ± 0.02 and 0.85 ± 0.04 µg, at 5 and 10 µg/ml treating concentrations, respectively), followed by NLb1 (0.24 ± 0.01 and 0.74 ± 0.02, at 5 and 10 µg/ml treating concentrations, respectively). The same situation as in hCMEC/D3, NLb2 was observed to have the lowest cellular uptake with 0.21 ± 0.01 and 0.72 ± 0.04 µg, at 5 and 10 µg/ml treating concentrations, respectively. Obtained results are in accordance with the literature data suggesting that non-PEGylated liposomes are prone to more efficient uptake by neuroblastoma cells. This outcome is probably due to the fact that PEG chains hinder the interactions of the liposomes with different membrane structures of this type of cells, thus resulting with poor intracellular transport [ 45 ]. On the other hand, neurons exhibit membranes which are unique in its composition being highly enriched in lipids, in particular cholesterol, which plays key role in regulation of the membrane structure, fluidity and permeability as well as multiple aspects of the synaptic transmission [ 46 ]. In pure human SH-SY5Y cell cultures, the glia-derived cholesterol is non-existing, and addition of cholesterol is needed in order to achieve conditions resembling normal neuronal environment with surrounding glial cells, as well as to promote the process of the SH-SY5Y neuroblastoma cell differentiation into neuronal cell type [ 47 ]. Up to date, several findings reported the clear preference of SH-SY5Y neurons for cholesterol containing liposomes. Namely, Lee et al. (2013) reported that the addition of cholesterol into the liposomal formulation resulted with 11-fold enhanced uptake by this cell culture line, implying on the fact that the composition of NLs significantly affects their uptake by neuronal cells and are avidly taken up by the addition of cholesterol [ 48 ]. In this sense, since all three nano-formulations contain cholesterol into their lipid bilayer, the lower uptake of the PEGylated liposomes (NLb1 and NLb2) may be a result of the steric effect of the PEG chains onto the surface, which probably act as a barrier and prevent the access of cholesterol to the cellular structures. It is also important to be mentioned that PEGylation can prevent or reduce, but does not totally exclude the protein binding to the NLs surface. Excessive PEGylation may contribute to less efficient binding with protein targets that would work as ligands for receptor mediated transport and delivery, finally resulting with partial inhibition and reduction of the cellular uptake (Pozzi et al., 2014). Taken into consideration all above mentioned, it can be summarized that cellular transport and internalization are influenced by numerous features such as the NLs composition (especially the amount of PEG), physico-chemical properties of NLs, the experimental conditions (concentration and incubation time), as well as the composition of the cell medium and the structural characteristics of cell culture lines. Cell uptake assessment of NLs in presence of transport pathways inhibitors In order to have insights into the mechanism of internalization of the NLs, as well as to better understand and correlate with the previously presented quantitative results for cell internalization at 37°C, uptake experiments in presence of specific inhibitors of endocytotic pathways were performed. In this sense, the cell culture lines (hCMEC/D3 and SH-SY5Y) were pretreated (40 minutes) with chlorpromazine or indomethacin as specific inhibitors of chlatrin and caveolin-mediated endocytosis. In addition, uptake experiments at 4°C were conducted, when it is supposed that all ATP-dependent transport mechanisms are blocked (Table S1 ). The fluorescence of cells incubated with NLs at 37°C was considered as 100%, while the fluorescence after incubation in the presence of inhibitors was expressed as a relative percentage compared to the cells without inhibitor. The statistical analysis of the obtained results for hCMEC/D3 cells (Fig. S5 a-c) clearly shows the concentration and endocytosis inhibitors having a significant effect, while lowering the temperature of the experiment (total energy metabolism) had no significant effect on the total uptake. In addition, the type of formulation, i.e. the amount of PEG on the NLs surface also affects the uptake, as seen on the VIP plot which provides an overall representation of the effect of the independent variables (Fig. S5 d). The SH-SY5Y uptake pattern showed a different trend of the influence of the independent variables (Fig. S6 a-c). According to the VIP plot (Fig. S6 d), it can be concluded that the concentration of the sample and also the temperature of the experiment are dominant factors affecting the uptake. Similarly, as with hCMEC/D3, type of formulation or more precisely, PEG amount also demonstrated a significant effect on the uptake under varying experimental conditions. From Fig. 6 can be observed that incubation at 4°C induces cell metabolic inhibition, resulting (for the concentration of 10 µg/ml) in a reduction of ~ 30% of the uptake of all formulations in both cell lines, compared to the experiments performed at 37°C. This indicates that energy-dependent endocytosis is included in the NLs uptake along with physical adhesion or passive diffusion [ 49 ]. Figure 6 In order to investigate the mechanism of endocytosis, cells were also treated with chlorpromazine which is known to inhibit AP2, one of the key adaptor proteins in clathrin-mediated endocytosis and it is also involved in clathrin accumulation in late endosomes, thereby inhibiting coated pit endocytosis. Figure 6 shows a decrease in the uptake of NLb1 and NLb2 by ∼25% in both cell lines compared to the control at 37°C, referring to the fact that chlatrin-mediated endocytosis may be involved, one of the predominant internalization pathways for the uptake of NPs ∼120 nm, because of the size of clathrin coated pits [ 49 – 51 ]. Additionally, the performed experiments resulted with significant reduction in the uptake of NLb0 in hCMEC/D3 (50.59 ± 2.65%), whereas only a slight decrease was observed in SH-SY5Y (93.78 ± 4.58%). This could be due to the different structural specificities and the distinct cell surface properties as well as the specific PC formed onto the surface of NLs after incubation with the cell culture medium [ 52 ]. On the other hand, it should be taken into consideration that when attempting to block a certain transport pathway, different types of cells usually adapt via activation of alternative mechanisms as well as overcompensation for the blocked function or receptor (Francia et al., 2019). This statement can further explain the heterogeneous results obtained for the inhibition of caveolin-mediated endocytosis with indomethacin between the different formulations in the different cell lines (49.96 ± 2.95–87.10 ± 3.56% and 57.17 ± 1.56–92.38 ± 2.65%, for hCMEC/D3 and SH-SY5Y, respectively). The surface properties of the nano-systems such as PEGylation can also affect the cell uptake/adhesion since PEG chains conformation and also, the aggregation of PEG polymers in the contact region between a PEGylated liposome and the membrane can influence the membrane wrapping process of PEGylated liposomes during endocytosis [ 53 ]. Therefore, it can be that different energy-dependent and non-dependent pathways are probably included in the dictation of NLs transport across BBB and neurons. Cell uptake experiments on co-cultured hCMEC/D3 and SH-SY5Y cell line Several studies have reported the internalization and uptake of NLs by different types of neuronal and BBB cell culture lines, individually. In this sense, detailed experiments were conducted under different experimental conditions on the two cell culture lines, hCMEC/D3 and SH-SY5Y, in order to determine the quantitative cell uptake and predict the internalization mechanism of the NLs investigated. However, despite the confirmed uptake of SH-SY5Y neuroblastoma cells after direct exposure to NLs presented earlier, it is not certain whether the results would be consistent and the obtained effects would be replicated in vivo , where the ability of nano-carriers to serve as platforms for active components intended for CNS treatment is limited due to the primary challenge of permeating across the BBB [ 54 ]. Another thing that should be taken into consideration is the fact that the information regarding the fate of the nano-systems in pericytes, astrocytes or neurons after having crossed the BBB is quite limited [ 55 ]. For this reason, the human derived brain endothelial cells, hCMEC/D3, were cultured on the apical side of permeable Transwell inserts, while monolayers of SH-SY5Y neuroblastoma cells were seeded on the basal side of 12-well plates chambers. After hCMEC/D3 reached a TEER value > 230 Ω and the confluence of SH-SY5Y was > 85%, both cell culture lines were combined and transport studies of the three NLs formulations were performed. The results from the NLs’ cellular uptake into neuronal cells after crossing the blood-brain barrier in vitro in our study suggest that the non-PEGylated formulation (NLb0) is internalized in highest percent (27.54 ± 2.93%), followed by NLb2 (26.46 ± 1.87%) and by the formulation with the highest amount of PEG onto its surface - NLb1 (25.17 ± 1.74%). This implies the successful transport of these liposomal nano-carriers across a BBB model and the consequent uptake of the particles by the neuronal cells [ 56 ]. It was also noticed that the quantitative uptake trend by SH-SY5Y in combination with hCMEC/D3 for the three different formulations is in good relation to the experiments on the single cell line (33.27 ± 1.95, 25.17 ± 2.65 and 26.46 ± 1.54% for NLb0, NLb1 and NLb2, respectively), which could be attributed to the physico-chemical properties of NLs and physiological factors affecting their internalization as well as the morphological properties of SH—SY5Y already discussed. Recent studies have highlighted the pivotal role of co-culture models in advancing in vitro neurotoxicity research. These models have significantly contributed to bridging the gap in faithfully replicating the human BBB phenotype. This fidelity is crucial for conducting permeability studies, assessing neurotoxicity, and investigating aspects related to neurodegenerative diseases [ 57 ]. The findings from the study of Freese et al. (2014) illustrates the effectiveness of a new hCMCEC/D3 – SH-SY5Y bio-assay in vitro system which proves adept at predicting drug penetration across the BBB, particularly for drugs relevant to Alzheimer's disease (AD) therapy [ 58 ]. Тhis same in vitro model with slight modifications was used by Mursaleen et al. (2021) in order to demonstrate that micellar nanocarriers loaded with hydroxytyrosol effectively crossed the BBB in vitro without inducing cytotoxicity. Moreover, these nanocarriers protected neuronal SH-SY5Y cells against rotenone-induced oxidative stress, as assessed by mitochondrial hydroxyl levels [ 56 ]. However, it is important to note that in this study, the evaluation of results was done based on the biological activity of the drug, not through measurement of the micelle carriers permeation and uptake by brain cells. When it comes to permeability studies of lipid nanoparticles with different surface characteristics across hCMEC/D3 and their subsequent uptake in SH-SY5Y, the literature is limited and generally focused on research involving ligand-functionalized lipid nano-systems. In this context, one of the few studies available is evaluation of the efficacy of apolipoprotein E (APOE) targeting nanoparticles for delivering donepezil across the BBB. The results underscored the effective permeability of targeting nanoparticles across the BBB and the findings indicated that nanoparticles equipped with APOE targeting ligand demonstrated higher cellular uptake compared to the non-functionalized ones [ 59 ]. Internalization studies To further verify the abovementioned results, the internalization of NLs by hCMEC/D3 and SH-SY5Y cell lines was investigated using fluorescent live-cell imaging and confocal microscopy. Figure 7 (a-c) and 8 (a-c) show images obtained by fluorescent microscopy of NLs incubated in both cell lines for 1, 2 and 4 h. From the microscopic images, the time-dependent internalization of all three formulations can be observed in both cell lines, where higher amounts of internalized nanoliposomal vesicles were noted in later time intervals. Additionally, from the fluorescence intensity, it can be seen that in the cells of the blood-brain barrier, the largest amount of internalized vesicles is attributed to NLb1, followed by NLb0 and NLb2, respectively, whereas in neuroblastoma cells, NLb0 exhibits the highest percentage of uptake, which is consistent with previous studies on the quantitative uptake at 37°C. Figure 7 Figure 8 Many research studies have demonstrated that liposomes tend to follow an endocytic mechanism of cellular internalization. Therefore, it was also important to visualize the NLs internalization pathway and confirm their co-localization in endosomes. In this direction, NLs were incubated for 4 h in the presence of a dye that signals endocytosis, i.e. in an acidic environment (endosomes and lysosomes) gives green fluorescence. From the presented images on Fig. 9 (a-c) it could be seen and confirmed that all formulations of NLs have been internalized and co-localized in the endosomal compartments in SH-SY5Y cells. Figure 9 From the images obtained by confocal microscopy, the internalization of NLs can be confirmed in hCMEC/D3 cell line. Literature data suggests on the ability of lipid NLs to cross BBB despite its highly restrictive nature, and moreover, deliver the encapsulated drugs in different cell compartments [ 60 ]. When it comes to nanoliposomes, as it could be seen from the presented images on Fig. 10 (a-c), they show tendency of accumulation around the perinuclear area. [ 61 ]. Regarding the intracellular localization of NLs, the obtained results showed that there is no difference in the cell distribution of the different NLs formulations, or more precisely, the presence and the amount of PEG on their surface did not influence the intracellular NLs co-localization (Fig. 10 ). Figure 10 Conclusion In this work three different nanoliposomal formulations with different PEG amounts on the surface were prepared and appropriately characterized in a biorelevant manner. The results from the stability studies of the tested formulations confirmed that after incubation in cell culture medium there were no changes in the mean z-size of the sample with the highest amount of PEG (NLb1) which also confirmed the stability of this formulation. Additionally, serum proteins were found to likely stabilize the PEG-free formulation (NLb0) in terms of preventing the aggregation process. Furthermore, by electrophoresis experiments, it was evident that protein corona was formed within the first hour of incubation in the serum supplemented culture medium, and the protein that was adsorbed in the largest percentage on the surface of NLs was albumin. Statistical analysis performed on the cell uptake pattern showed that NLs concentration and incubation time play a key role on the percentage of internalized NLs. Furthermore, the highest uptake by hCMEC/D3 cell line was obtained for the formulation with the highest amount of PEG on the surface (NLb1). A different situation was observed for the cellular uptake by SH-SY5Y, where the PEG-free formulation (NLb0) gave the most successful internalization. When it comes to the mechanism of cellular internalization, all nano-vesicle samples were characterized by energy-dependent endocytic transport and passive diffusion. The transport studies on the combined hCMEC/D3/SH-SY5Y cell line confirmed the successful transport of the nanoformulations across the BBB and their subsequent uptake by the neuroblastoma cells. The obtained micrographs from the fluorescent microscopy on live cells and the confocal microscopy gave insight into the successful internalization of the NLs in the BBB and neuroblastoma cells, in addition revealing that the co-localization of the NLs was in the perinuclear cell regions. From the above-mentioned, it can be concluded that all properties and performances of the designed NLs are in favor of the efficient brain delivery, and hence their potential for treatment of different CNS diseases. Declarations Ethics approval and Consent to participate This is an in vitro study and no human participants were involved, neither their data or biological material. Consent for publication In this study no human participants were involved, neither their data or biological material. Therefore, consent for publication is not available. Competing interests The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Funding Experimental data were in part generated under the Framework for Access to the Joint Research Centre Physical Research Infrastructures of the European Commission (Project: Proteomic profiling of the protein corona formed onto nanoparticles’ surface upon their exposure in HCMEC/D3 cell culture medium (PPPCNCCM), Research Infrastructure Access Agreement N° 36025/9; Call 2020-1-RD-Nanobiotech) and at the Department of Pharmaceutical Technology and Biopharmacy, Institute of Pharmaceutical Sciences at the University of Graz, Austria, as part of the CEEPUS student mobility program, through the CEKA PharmTech network (CIII-RS-1113-02-1819-Central European Knowledge Alliance for Teaching, Learning and Research in Pharmaceutical Technology). Authors’ contributions All authors contributed to the study conception and design. Investigation, experimental work, data analysis and interpretation were performed by Dushko Shalabalija and Ljubica Mihailova. The first draft of the manuscript was written by Dushko Shalabalija and Ljubica Mihailova and all authors commented on previous versions of the manuscript. Nikola Geskovski, Otmar Geiss, Sabrina Gioria and Diletta Scaccabarozzi were actively involved in the investigation, data analysis as well as writing and reviewing the manuscript. Andreas Zimmer and Marija Glavas Dodov besides the study conception and design, they also contributed with data analysis, reviewing and editing as well as supervision of the research activities. All authors read and approved the final manuscript. Data Availability Statement The datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request. 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Francia V, Reker-Smit C, Boel G, Salvati A. Limits and challenges in using transport inhibitors to characterize how nano-sized drug carriers enter cells. Nanomed. 2019;14(12):1533–49. Ou YH, Liang J, Chng WH, Muthuramalingam RPK, Ng ZX, Lee CK, et al. Investigations on Cellular Uptake Mechanisms and Immunogenicity Profile of Novel Bio-Hybrid Nanovesicles. Pharmaceutics. 2022;14(8):1738. Nie Y, Ji L, Ding H, Xie L, Li L, He B, et al. Cholesterol Derivatives Based Charged Liposomes for Doxorubicin Delivery: Preparation, In Vitro and In Vivo Characterization. Theranostics. 2012;2(11):1092–103. Shen Z, Ye H, Kröger M, Li Y. Aggregation of polyethylene glycol polymers suppresses receptor-mediated endocytosis of PEGylated liposomes. Nanoscale. 2018;10(9):4545–60. Mursaleen L, Chan SHY, Noble B, Somavarapu S, Zariwala MG. Curcumin and N-Acetylcysteine Nanocarriers Alone or Combined with Deferoxamine Target the Mitochondria and Protect against Neurotoxicity and Oxidative Stress in a Co-Culture Model of Parkinson’s Disease. Antioxidants. 2023 Jan;12(1):130. Porkoláb G, Mészáros M, Tóth A, Szecskó A, Harazin A, Szegletes Z, et al. Combination of Alanine and Glutathione as Targeting Ligands of Nanoparticles Enhances Cargo Delivery into the Cells of the Neurovascular Unit. Pharmaceutics. 2020;12(7):635. Mursaleen L, Noble B, Somavarapu S, Zariwala MG. Micellar Nanocarriers of Hydroxytyrosol Are Protective against Parkinson’s Related Oxidative Stress in an In Vitro hCMEC/D3-SH-SY5Y Co-Culture System. Antioxid Basel Switz. 2021;10(6):887. Monteiro AR, Barbosa DJ, Remião F, Silva R. Co-Culture Models: Key Players in In Vitro Neurotoxicity, Neurodegeneration and BBB Modeling Studies. Biomedicines. 2024;12(3):626. Freese C, Reinhardt S, Hefner G, Unger RE, Kirkpatrick CJ, Endres K. A Novel Blood-Brain Barrier Co-Culture System for Drug Targeting of Alzheimer’s Disease: Establishment by Using Acitretin as a Model Drug. Iijima KM, editor. PLoS ONE. 2014;9(3):e91003. Topal GR, Mészáros M, Porkoláb G, Szecskó A, Polgár TF, Siklós L, et al. ApoE-Targeting Increases the Transfer of Solid Lipid Nanoparticles with Donepezil Cargo across a Culture Model of the Blood–Brain Barrier. Pharmaceutics. 2020;13(1):38. Reginald-Opara JN, Svirskis D, Paek SY, Tang M, O’Carroll SJ, Dean JM, et al. The involvement of extracellular vesicles in the transcytosis of nanoliposomes through brain endothelial cells, and the impact of liposomal pH-sensitivity. Mater Today Bio. 2022;13:100212. Boado RJ, Pardridge WM. The Trojan Horse Liposome Technology for Nonviral Gene Transfer across the Blood-Brain Barrier. J Drug Deliv. 2011;2011:296151. Supplementary Files GA.png Graphical Abstract SupplementaryMaterial.docx Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4828653","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":338655418,"identity":"641b6e6a-f462-4dd2-9593-3673fb281db0","order_by":0,"name":"Dushko 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Pharmacy","correspondingAuthor":false,"prefix":"","firstName":"Marija","middleName":"Glavas","lastName":"Dodov","suffix":""}],"badges":[],"createdAt":"2024-07-30 12:07:13","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4828653/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4828653/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":64095210,"identity":"818706e8-7041-471e-bb29-0d1dbf74be13","added_by":"auto","created_at":"2024-09-06 16:49:45","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":298720,"visible":true,"origin":"","legend":"\u003cp\u003eFractograms from the qualitative evaluation using AF4-UV-MALS-DLS of the native formulations a) NLb0, b) NLb1, c) NLb2\u003c/p\u003e","description":"","filename":"1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4828653/v1/74e631c87c9474e03837b77d.jpg"},{"id":64095219,"identity":"c3114cad-3484-4769-9510-b742c46416f9","added_by":"auto","created_at":"2024-09-06 16:49:46","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":177049,"visible":true,"origin":"","legend":"\u003cp\u003eComparative representation of the UV signal and z-average diameter of NLb1 after a) 1 h incubation in serum supplemented (S) and serum free (M) cell culture medium, b) 4 h incubation in serum supplemented and serum free cell culture medium, c) 1 and 4 h of incubation in serum free cell culture medium, d) 1 and 4 h incubation in serum supplemented cell culture medium\u003c/p\u003e","description":"","filename":"Fig.2.png","url":"https://assets-eu.researchsquare.com/files/rs-4828653/v1/64c44adf6038ab82a5d36bc1.png"},{"id":64095211,"identity":"2af99bc6-147e-40e6-965e-e9228321cf12","added_by":"auto","created_at":"2024-09-06 16:49:46","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":155434,"visible":true,"origin":"","legend":"\u003cp\u003eComparative representation of the UV signal and z-average diameter of NLb2 after a) 1 h incubation in serum supplemented (S) and serum free (M) cell culture medium, b) 1 and 4 h incubation in serum supplemented cell culture medium, c) 4 h incubation in serum supplemented and serum free cell culture medium, d) 1 and 4 h of incubation in serum free cell culture medium\u003c/p\u003e","description":"","filename":"Fig.3.png","url":"https://assets-eu.researchsquare.com/files/rs-4828653/v1/2ce27846017fd94a10118132.png"},{"id":64095541,"identity":"b3e7f764-c90d-4980-973b-a4f245af6f03","added_by":"auto","created_at":"2024-09-06 16:57:46","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":157626,"visible":true,"origin":"","legend":"\u003cp\u003eComparative representation of the UV signal and z-average diameter of NLb0 after a) 1 h incubation in serum supplemented (S) and serum free (M) cell culture medium, b) 1 and 4 h incubation in serum supplemented cell culture medium, c) 4 h incubation in serum supplemented and serum free cell culture medium, d) 1 and 4 h of incubation in serum free cell culture medium.\u003c/p\u003e","description":"","filename":"Fig.4.png","url":"https://assets-eu.researchsquare.com/files/rs-4828653/v1/faa1ed490885c85221cd95c3.png"},{"id":64095214,"identity":"bcee140b-3a90-4909-9ee2-489a541b9b01","added_by":"auto","created_at":"2024-09-06 16:49:46","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":245960,"visible":true,"origin":"","legend":"\u003cp\u003eHigh-resolution automated electrophoresis band representation of a) NLb1 and NLb2 (labelled as Material 1 and 2, respectively) after 1 and 4 h incubation in serum free cell medium (M) and serum supplemented cell medium (S), b) NLb0 (labelled as Material 3) after 1 and 4 h incubation in serum-free cell medium (M) and serum-supplemented cell medium (S)\u003c/p\u003e","description":"","filename":"5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4828653/v1/f09b7587fc84744f77af7d9f.jpg"},{"id":64095542,"identity":"2e2efcef-0154-4c6b-a37f-ce3d3593d183","added_by":"auto","created_at":"2024-09-06 16:57:46","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":262352,"visible":true,"origin":"","legend":"\u003cp\u003eCell uptake of NLs (10 μg/mL) in a) hCMEC/D3 cells; b) SH-SY5Y after 2 h incubation at 4 °C, with chlorpromazine or indomethacin\u003c/p\u003e","description":"","filename":"6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4828653/v1/7e13e698a94d70c22181a93f.jpg"},{"id":64095216,"identity":"6403fc2e-e095-485d-b1db-c7adea7f9174","added_by":"auto","created_at":"2024-09-06 16:49:46","extension":"jpg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":298604,"visible":true,"origin":"","legend":"\u003cp\u003eInternalization in live hCMEC/D3 cells of a) NLb0; b) NLb1; c) NLb2 by fluorescent microscopy (Nile-Red channel used)\u003c/p\u003e","description":"","filename":"7.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4828653/v1/2486918d30022d2f0766554c.jpg"},{"id":64095217,"identity":"4ef6731c-4e9e-4638-9b9f-be60a5d57fc1","added_by":"auto","created_at":"2024-09-06 16:49:46","extension":"jpg","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":322852,"visible":true,"origin":"","legend":"\u003cp\u003eInternalization in live SH-SY5Y cells of a) NLb0; b) NLb1; c) NLb2 by fluorescent microscopy (Nile-Red channel used)\u003c/p\u003e","description":"","filename":"8.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4828653/v1/dfd4101fb7dd7e0f581f269d.jpg"},{"id":64095221,"identity":"0c99e0fc-58f8-4387-88cd-f55befc407b5","added_by":"auto","created_at":"2024-09-06 16:49:47","extension":"jpg","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":322452,"visible":true,"origin":"","legend":"\u003cp\u003eRepresentation of a) NLb0, b) NLb1 and c) NLb2, in live SH-SY5Y cells (4 h) by fluorescent microscopy (Left image – phase contrast; Second image – red fluorescence by NLs marked with Dil red; Third image – green fluorescence by endosomes marked with pHrodo Green dextran conjugate, Right image – superimposed image)\u003c/p\u003e","description":"","filename":"9.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4828653/v1/330ee7bf4a6cc8cb8dcbbaed.jpg"},{"id":64095218,"identity":"e3b17ba0-29ee-4956-ba79-e3b02521d1ca","added_by":"auto","created_at":"2024-09-06 16:49:46","extension":"jpg","order_by":10,"title":"Figure 10","display":"","copyAsset":false,"role":"figure","size":778554,"visible":true,"origin":"","legend":"\u003cp\u003eConfocal microscopy of internalization and distribution of a) NLb0, b) NLb1 and c) NLb2 in hCMEC/D3\u003cem\u003e \u003c/em\u003ecell line. Upper left image - blue channel – nucleus counterstained with Hoechst Fluorescent stain and DAPI excited at 405 nm and detected by bandpass filter (BP 420/480 nm); Upper right - green channel - actin cytoskeleton stained with Alexa Fluor 488 Phalloidin excited at 488 nm and detected by bandpass filter (BP 505/550 nm); Bottom left image - red channel - Dil-labelled samples detected at a 549 nm excitation wavelength by longpass filter (LP 560 nm); Bottom right image – superimposed micrograph)\u003c/p\u003e","description":"","filename":"10.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4828653/v1/75cd51721b806779b1ccd3e7.jpg"},{"id":74189449,"identity":"d75a1117-239b-430d-a024-df814ad9ce8a","added_by":"auto","created_at":"2025-01-19 18:14:38","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":4096952,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4828653/v1/d91a5fc9-4a76-4f63-bd52-d022db112f28.pdf"},{"id":64095540,"identity":"faf46f69-d31e-485b-8c92-3c07604f75ea","added_by":"auto","created_at":"2024-09-06 16:57:45","extension":"png","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":189776,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eGraphical Abstract\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"GA.png","url":"https://assets-eu.researchsquare.com/files/rs-4828653/v1/8badb538c2a2ce2ffb6c3caf.png"},{"id":64095222,"identity":"45816fa7-71f0-4f0c-b929-3c4607003937","added_by":"auto","created_at":"2024-09-06 16:49:48","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":920775,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryMaterial.docx","url":"https://assets-eu.researchsquare.com/files/rs-4828653/v1/9edcb3344e9f676fb74e1292.docx"}],"financialInterests":"","formattedTitle":"Influence of surface characteristics on the in vitro stability and cell uptake of nanoliposomes for brain targeting","fulltext":[{"header":"Introduction","content":"\u003cp\u003eAdvancements in nanotechnology, coupled with the recognition of the benefits of utilizing nanoliposomes (NLs) as carriers for targeted delivery and controlled release of active components, suggest the use of these multifunctional platforms to address multiple pathologies associated with neurodegenerative diseases, such as Alzheimer's disease (AD), consequently impeding their progression [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Therefore, incorporating active components into these vesicles enhances their biological distribution, diminishes macrophage uptake in the reticuloendothelial system, and also lowers free drug concentrations, thus leading to a decrease in systemic toxicity. Adjustments to the composition and surface characteristics of NLs hold the potential for facilitating effective passage through the blood-brain barrier (BBB) as well as specific targeting of various brain structures [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eHowever, despite the wide spectrum of advantages, only a limited number of NLs formulations designed for the treatment of brain diseases have successfully undergone the clinical evaluation and are currently available on the market [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. This limitation could be attributed to a lack of comprehensive understanding of the crucial factors influencing the optimal delivery of active components to the central nervous system (CNS), particularly in the early stages of the development process. Furthermore, gaining a detailed insight and comprehension of the mechanisms governing the transport of NLs across the blood-brain barrier (BBB), along with the subsequent changes occurring during the delivery of active components to the brain, is imperative. Moreover, it is essential to discover how these kinetic processes are influenced not only by the properties of NLs formulations but also by the pathophysiology of the condition [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eTaking into consideration the clinical potential of NLs as nano-carriers, European Medicines Agency (EMA) and U.S. Food and Drug Administration (FDA) issued guidances for the development of liposomal drug products, outlining several critical quality attributes (CQAs) that should be taken into consideration during the formulation and manufacturing stages. These CQAs encompass: identification and quantification of lipid composition; quantification of encapsulated, free, and total active component; characterization of NLs in terms of morphology, structure, particle size distribution, and surface charge; assess of the physical (fusion and aggregation) and chemical (lipid and active component degradation) stability and evaluation of the \u003cem\u003ein vitro\u003c/em\u003e drug release kinetics [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eRegarding the aforementioned part, in the contemporary research, it is evident that there is a significant emphasis on the physicochemical characteristics and efficiency of NLs formulations. However, minimal research has been dedicated to elucidating nano-bio interactions and comprehending the behavior of these formulations at organ and cellular levels. Namely, it is well known that, upon exposure to biological fluids, nano-delivery systems, including NLs, swiftly engage with various biomolecules, primarily involving three key aspects: (1) the adsorption of biomolecules onto the surface of nano-systems, leading to the formation of a protein corona (PC); (2) the reconstruction and alteration of functional proteins; and (3) redox reactions occurring between nano-systems and reactive species [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. These interactions between nanomaterials and biological entities not only significantly impact the functionality and fate of the nano-systems but also influence cellular biological functions and may affect their targeting yields, the bio-distribution and cell internalization [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. The current components of the serum could potentially exert significant deleterious effects on the lipid NLs structure, leading to the disruption of lipid bilayers and subsequent leakage of the encapsulated contents [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Conversely, research indicates that the adherence of plasma proteins to the surface of NLs may facilitate particle aggregation. As PC accumulates on the NLs' surface, it alters their surface characteristics. Consequently, the presence of proteins in the tissue milieu could potentially modify the cellular uptake mechanisms of both cationic and anionic nanocarriers. [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. In simpler terms, our understanding of what precisely occurs to nano-delivery systems in a biological environment is limited. Questions linger regarding potential changes, such as alterations in their surface area, and how these modifications impact their interactions within the biological matrix. Therefore, of outmost importance is to assess the fundamental mechanisms governing reactions between the nano-carrier systems and the bioenvironmental components (nano-bio) in direction of devising strategies for manipulating these nano-bio reactions.\u003c/p\u003e \u003cp\u003eIn this context, monitoring the stability of nano-carriers in biological environments can be effectively achieved using advanced techniques such as asymmetric flow field-flow fractionation (AF4). AF4 has gained recognition as a crucial method for characterizing parameters such as particle size, polydispersity, drug loading, and overall stability of nanoparticle dispersions, covering a broad spectrum of particle sizes and sample polydispersity [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. In recent years, collaborative efforts between the European Nanomedicine Characterization Laboratory (EUNCL) and the National Cancer Institute Nanotechnology Characterization Laboratory (NCI-NCL) have been pivotal. These collaborations have focused on developing robust Standard Operating Procedures (SOPs) tailored for measuring the physico-chemical properties of nanopharmaceuticals. This includes lipid-based nanocarriers such as liposomes and other (phospho)lipid nanoparticles, ensuring consistent and reliable characterization across different research settings and applications [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn addition to the aforementioned studies on the modified NLs surface, \u003cem\u003ein vitro\u003c/em\u003e cell culture models offer a valuable avenue for gaining a deeper understanding of complex human organs like the brain and enhancing the translational relevance of \u003cem\u003ein vivo\u003c/em\u003e models. Optimal selection of cell lines and fine-tuning experimental conditions serve as promising tools for comprehending the behavior of nano-delivery systems (NDS) upon contact with biological fluids and the microenvironment. Furthermore, these approaches facilitate the prediction of NDS' \u003cem\u003ein vivo\u003c/em\u003e stability, toxicity, and therapeutic potential [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. In this direction, hCMEC/D3 cell culture line is one of the \u003cem\u003ein vitro\u003c/em\u003e models providing detailed insights into the uptake and transport of novel drug candidates and NDS across the BBB, influencing their subsequent therapeutic efficacy in the brain [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. This model boasts advantages such as easy cell growth, mimicking basic BBB properties and morphological characteristics even without co-cultured glial cells [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. Nonetheless, a major challenge lies in optimizing the tightness of tight junctions in cell monolayers to create a relevant \u003cem\u003ein vitro\u003c/em\u003e model that mirrors human BBB characteristics, including permeability restriction with functional efflux and influx transporters and molecular exclusion [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. Another relevant \u003cem\u003ein vitro\u003c/em\u003e cell model used for predicting the \u003cem\u003ein vivo\u003c/em\u003e behavior, internalization and performances of NDS in the brain tissues upon their successful transport across the BBB is the SH-SY5Y human neuroblastoma cell line which is characterized by rapid proliferation and non-expression of mature neuronal markers [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. In this sense, this cell line can further be differentiated into neuronal like-morphologies by well-established protocols [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. Similarly, as hCMEC/D3, undifferentiated SH-SY5Y cell culture line has been used as \u003cem\u003ein vitro\u003c/em\u003e model for evaluation of the effect of neurotoxins as well as for investigation of NDS uptake and drug efficacy in the treatment of Alzheimer\u0026rsquo;s and Parkinson\u0026rsquo;s disease (Riegerov\u0026aacute; et al., 2021). This immature population of cells proliferate continuously as adherent catecholaminergic neuroblasts with a low portion of epithelial-like cells. It is important to mention that they do not possess the morphological, biochemical and functional properties of mature neurons [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIt is important to emphasize that literature data on physical stability of NLs in different physiological barriers and compartments is limited. Therefore, the main aim of this research was to shed light on certain segments in the behavior of these carriers in a research that is designed in such a way to examine the evolution of the physico-chemical characteristics and their surface in biorelevant media.\u003c/p\u003e \u003cp\u003eAdditionally, the interaction of these carriers with relevant cell types under various conditions was examined to further evaluate whether the physico-chemical properties and stability of the prepared formulations affects their performance in an \u003cem\u003ein vitro\u003c/em\u003e cell culture environment. For this purpose, detailed time- and concentration-dependent uptake experiments were carried out on hCMED/D3 and SH-SY5Y cell lines, separately, in order to evaluate the influence of the formulation properties as well as the extracellular microenvironment on the NLs internalization kinetics and mechanism in the brain tissues. A step forward was also made by conducting transport experiments on a co-culture system (hCMED/D3 / SH-SY5Y) with the aim to confirm the efficient transit of NLs across the BBB and their subsequent successful uptake into the neurons.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eMaterials\u003c/h2\u003e \u003cp\u003eSoybean lecithin (SL) was purchased from Vitalia (Skopje, N. Macedonia) and LIPOID PE 18:0/18:0-PEG 2000 (PEG) from Lipoid (Ludwigshafen am Rhein, Germany). Cholesterol (CH), Dulbecco\u0026rsquo;s Phosphate Buffered Saline (DPBS), collagen type I from rat tail, fluorescein isothiocyanate isomer I (FITC) and Nile Red dye were obtained from Sigma Aldrich (St. Louis, USA). Immortalized human cerebral microvascular endothelial (hCMEC/D3) cell line (CELLutions, Biosistems/Cedarlane\u0026reg;, Canada) were maintained in Endothelial Basal Medium-2 (EBM-2), supplemented with Fetal Bovine Serum (FBS), chemically defined lipid concentrate, HEPES 1M and penicillin\u0026thinsp;\u0026minus;\u0026thinsp;streptomycin (Life Technologies, California, USA), human basic Fibroblast Growth Factor (bFGF), ascorbic acid and hydrocortisone (Sigma Aldrich, St. Louis, USA) and detached with trypsin-EDTA (GIBCO, Thermo Fisher Scientific, California, USA). Human neuroblastoma cell line (SH-SY5Y) was purchased from LCG Standards (Wesel, Germany) and maintained in Dulbecco's Modified Eagle Medium (DMEM) (Thermo Fisher Scientific, California, USA). EndoGRO-MV SCME004 complete media kit was provided by Merck (Merck Group, Germany). CellTiter 96 AQueous Non-Radioactive Cell Proliferation Assay (MTS) and CytoTox-ONETM Homogeneous Membrane Integrity Assay (CytoTox) were obtained by Promega (Wisconsin, USA). Alexa FluorTM Phalloidin 488, Hoechst Fluorescent stain and Dil stain (1,1'-Dioctadecyl-3,3,3',3'-Tetramethylindocarbocyanine Perchlorate ('DiI'; DiIC18(3))) were purchased from Thermo Fisher Scientific (California, USA).\u003c/p\u003e \u003cp\u003eAll chemicals and reagents used were of the highest purity grade commercially available.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003ePreparation of the nanoliposomes (NLs)\u003c/h2\u003e \u003cp\u003eNLs samples (SL:CH:PEG\u0026thinsp;=\u0026thinsp;8.71:1:0; 8.71:1:1.67 and 8.71:1:0.67 molar ratios for NLb0, NLb1 and NLb2, respectively) were prepared by modified lipid film hydration technique as described by Shalabalija et al. (2021). All fluorescent dyes, Nile Red (1.6%, \u003cem\u003ew/w\u003c/em\u003e) and DIl stain (2.5%, \u003cem\u003ew/w\u003c/em\u003e), used for the purpose of the cell culture experiments were added in the organic phase during the preparation process according to Mihailova et al. (2023).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eParticle size, particle size distribution and z-potential of the NLs\u003c/h2\u003e \u003cp\u003eТhe z- average diameter, the polydispersity index (PDI) and z-potential (ZP) were determined using a Zetasizer Nano Series, Nano-ZS, (Malvern Instruments Ltd., UK), after diluting the optimal NLs samples in 10 mM PB pH 7.4 (1:20, \u003cem\u003ev/v\u003c/em\u003e). Measurements were made under the following conditions: 25\u0026deg;C, thermostating time of 120 seconds, viscosity of the medium 0.8894 cP, dielectric constant 78.5 and an angle of 173\u0026deg;. At least 3 separate preparations (batches) from each sample were measured in triplicate. Results of each analysis was the average of 12 consecutive measurements.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eStability studies of NLs in cell culture medium\u003c/h2\u003e \u003cp\u003eThe samples (NLb0, NLb1 and NLb2) at a native concentration of 30 mg/ml were firstly diluted to a final concentration of 1 mg/ml (total volume of 2 ml) in cell culture medium (Endo-GRO-MV, Cat SCME004, Sigma Aldrich), and then incubated at 37\u0026deg;C for 1 and 4 h in an Eppendorf thermomixer under constant stirring (300 rpm). Incubation took place in serum free and 5% serum supplemented cell culture medium (prepared according to the manufacturer's instructions). At the end of the incubation time, the samples were vortexed (5 seconds) and a volume of 1 ml was transferred to a vial for asymmetric flow field-flow fractionation analysis (AF4 analysis). Experiments were carried out by in-line coupling the AF4 system with UV/VIS, MALS and DLS detectors.\u003c/p\u003e \u003cp\u003eThe AF4 system used was composed of an Eclipse Dualtec separation system (Wyatt Technology Europe GmbH, Dernbach, Germany) and an Agilent 1260 Infinity high-performance liquid chromatograph (Agilent Technologies, Santa Clara, USA) equipped with a degasser (G1322A), an isocratic pump (G1310B), autosampler (G1329B) and multiple wavelength (MWD) detector (G1365C) ​​set at 230 nm. In the Eclipse SC separation channel, regenerated cellulose membranes (10 kDa) and a spacer height of 350 \u0026micro;m were used. Phosphate buffer (PBS) was used as eluent. The detector flow rate was set at 0.5 ml/min and the injection volume was 50 \u0026micro;l. Separation settings were: a) Elution 0\u0026ndash;3 mins; b) Focus: 3\u0026ndash;5 mins (Focus flow: 1.0 ml/min); c) Focus\u0026thinsp;+\u0026thinsp;Inject: 5\u0026ndash;10 mins (Focus flow: 1.0 ml/min); d) Elution 10\u0026ndash;50 mins (Cross-flow from 1.0\u0026ndash;0.1 ml/min); e) Elution 50\u0026ndash;55 mins (Cross-flow 0.1 ml/min); f) Elution 55\u0026ndash;60 mins (Cross-flow 0.1\u0026ndash;0.0 ml/min); g) Elution: 60\u0026ndash;70 mins (Cross flow 0.0 ml/min). The outlet of the MWD detector was connected to a DAWN 8\u0026thinsp;+\u0026thinsp;HELEOS II multi-angle light scattering detector (MALS) operating with a 658 nm laser (Wyatt Technology Europe). A DLS (Malvern Zetasizer Nano-S, UK) with an installed quartz flow-cell (ZEN0023) was also used in this study in flow mode. Refractive index (RI) and absorption parameters were set to 1.38 and 0.010 respectively. Water was set as the dispersant, the temperature was set to 25\u0026deg;C, while the attenuation was set to 11. 'General purpose (normal distribution)' was chosen as the analysis model.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eProteomic profiling of the adsorbed proteins onto NLs surface\u003c/h2\u003e \u003cp\u003eProtein electrophoresis was performed by 2100 Bioanalyzer using High Sensitivity Protein 250 kit assays in reducing condition according to the manufacturer\u0026rsquo;s instruction. Briefly, samples prepared as for the AF4 analysis, at the end of the incubation time, were equilibrated using the standard labelling buffer and labelled fluorescent dye. Next, samples were diluted 200-fold in milliQ-water and denatured at 95\u0026deg;C for 5 min in reducing condition by adding 3.5 \u0026micro;l of 1 M Dithiothreitol (DTT) buffer solution to 100 \u0026micro;l of each sample buffer. After cooling, samples were loaded on the microfluidic chip for electrophoresis, in accordance with the manufacturer's instructions. All reagents and instruments were from Agilent Technology, California, USA.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003ehCMEC/D3 and SH-SY5Y cell culture lines\u003c/h2\u003e \u003cp\u003eBoth cell culture lines were seeded and cultivated according to the Supplier\u0026rsquo;s guidelines, explained in details in Mihailova et al. (2023).\u003c/p\u003e \u003cp\u003e \u003cem\u003eIn vitro\u003c/em\u003e uptake and internalization experiments were conducted using hCMEC/D3 cell cultures. T-75 cell culture flasks from Greiner Bio-One GmbH, Germany, were initially coated with 0.05 mg/ml rat tail collagen type I in DPBS and left to stand for at least 1 hour at 37\u0026deg;C. The cells were cultured in supplemented EBM-2 at 37\u0026deg;C with 5% CO\u003csub\u003e2\u003c/sub\u003e. Medium replacement occurred every 2\u0026ndash;3 days until cells reached confluence. Upon reaching confluence, the medium was aspirated, and cells were detached from the flask walls by incubating them at 37\u0026deg;C for 8 minutes in 0.1 mg/ml trypsin-EDTA solution. The cell suspension was then centrifuged at 1500 rpm for 3 minutes, and the supernatant was discarded. The cells were resuspended in 5 ml of cell medium and were prepared for further experimentation.\u003c/p\u003e \u003cp\u003eThe human neuroblastoma cell line was cultured in DMEM at 37\u0026deg;C in a 5% CO\u003csub\u003e2\u003c/sub\u003e atmosphere. Medium replacement occurred every 2\u0026ndash;3 days until cells reached confluence. During the splitting process, the medium was aspirated, and cells were detached from the flask walls after incubating at 37\u0026deg;C for 3 minutes in 0.1 mg/ml trypsin-EDTA solution. The cell suspension was then centrifuged at 800 rpm for 5 minutes, and the supernatant was discarded. The cells were subsequently resuspended in 5 ml of cell medium, making them ready for further experiments.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eCell uptake assessment of NLs\u003c/h2\u003e \u003cp\u003eIn order to determine the influence of the PEG amount and exposure times on the uptake of the NLs, hCMEC/D3 or SH-SY5Y cells were seeded in 96-well plates at a density of 10\u003csup\u003e4\u003c/sup\u003e cells/well in their respective cell culture medium (200 \u0026micro;l/well) and incubated for 48 h at 37\u0026deg;C and in the presence of 5% CO\u003csub\u003e2\u003c/sub\u003e. As previously described, wells for hCMEC/D3 cells were pre-coated with collagen type 1 (0.05 mg/ml) for 1 h. After reaching confluence, the cell culture medium was replaced and the cells were treated with different concentrations of the fluorescent dye labeled formulations NLb0, NLb1 and NLb2 with Nile Red (Sigma Aldrich, USA) (previously diluted with PBS and dispersed in the appropriate cell culture medium at final concentrations of 5, 10 or 100 \u0026micro;g/ml). Incubation was performed for 1, 2, and 4 h (37\u0026deg;C, 5% CO\u003csub\u003e2\u003c/sub\u003e), followed by washing step with PBS and lysis with 2% Triton X-100 (2 h, 37\u0026deg;C, 5% CO\u003csub\u003e2\u003c/sub\u003e). The resulting fluorescence was measured on a plate reader at 535 nm excitation and 635 nm emission (BMG Labtech, Ortenberg, Germany). Untreated cells were used as blank. The quantitative amount of internalized NLs was calculated according to pre-obtained regression analysis equations constructed from the fluorescence obtained over a range of concentrations for each of the formulations (non-incubated with cells). The experiments were performed three times on at least six replicates from each sample.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eCell uptake assessment of NLs in presence of transport pathways inhibitors\u003c/h2\u003e \u003cp\u003eIn order to determine the exact mechanism of cellular internalization, uptake experiments were also performed in the presence of inhibitors for specific transport pathways. Cells were seeded and cultured as described above.\u003c/p\u003e \u003cp\u003eThe quantitative amount of the internalized NLs was investigated on both cell lines. One set of cells was treated with 15 \u0026micro;M chlorpromazine and another set with 25 \u0026micro;M indomethacin for 40 min, followed by incubation with NLs (at final concentrations of 5, 10 or 100 \u0026micro;g/ml) for 2 h (37\u0026deg;C, 5% CO\u003csub\u003e2\u003c/sub\u003e). Third set of cells was left at 4\u0026deg;C for 40 min, before NLs incubation in cold condition for 2 h. The resulting fluorescence was measured as described previously (same conditions as for the 37\u0026deg;C experiments), and the quantitative amount of internalized NLs was calculated and expressed as the % of the uptake of the corresponding NLs concentrations at 37\u0026deg;C (taken as 100%). The experiments were performed three times on six replicates from each sample.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e1.1. Cell uptake experiments on co-cultured hCMEC/D3 and SH-SY5Y cell line\u003c/h2\u003e \u003cp\u003eThe hCMEC/D3 cells were seeded on Transwell inserts (5x10\u003csup\u003e4\u003c/sup\u003e cells/insert), previously coated with 0.05 mg/ml type I collagen in DPBS (1 h, 37 \u003csup\u003eo\u003c/sup\u003eC) and incubated with EBM-2 (0.5 ml cell medium in the apical part of the insert and 1 ml in the basal part of the well). The medium was changed every 2\u0026ndash;3 days until a transendothelial resistance (TEER) value\u0026thinsp;\u0026gt;\u0026thinsp;230 Ω was reached, indicative of solid monolayer formation and tight junctions between endothelial cells [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. In parallel, SH-SY5Y cells were seeded in 12-well plates (3x10\u003csup\u003e4\u003c/sup\u003e cells/well) and cultured in 1 ml DMEM for the same period of time, until reaching confluence. Furthermore, the inserts with hCMEC/D3 cells were placed in the plates with formed monolayers of SH-SY5Y. NLs previously diluted in PBS and dispersed in EBM-2 (final concentration 10 \u0026micro;g/ml) were added to the apical part of the inserts, and 1 ml of DMEM was added to the basal part of the plates. After 2 h of incubation (TEER\u0026thinsp;\u0026gt;\u0026thinsp;210 Ω), the inserts were removed and the SH-SY5Y wells were washed twice with PBS and lysed with 2% Triton X-100 (2 h, 37\u0026deg;C, 5% CO\u003csub\u003e2\u003c/sub\u003e). The resulting fluorescence was measured on a plate reader at 535 nm excitation and 635 nm emission (BMG Labtech, Ortenberg, Germany). The wells with SH-SY5Y incubated in the corresponding cell medium (not treated with NLs) were used as blanks. The quantitative amount of NLs taken up was calculated as % of the fluorescence of the native nano-formulations (in the same concentrations). The experiments were performed three times on at least six replicates from each sample.\u003c/p\u003e \u003cp\u003e \u003cem\u003eInternalization studies\u003c/em\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003ea) Internalization studies of NLs in live hCMEC/D3 and SH-SY5Y cell lines by fluorescence microscopy\u003c/h2\u003e \u003cp\u003eIn order to obtain a deeper insight of the internalization and co-localization of the prepared NLs samples in living cells, hCMEC/D3 and SH-SY5Y were first seeded on 35-mm glass dishes at a density of 2x10\u003csup\u003e5\u003c/sup\u003e cells per well (\u0026micro;-Dish 35 mm, WillCo Glass Bottom Dishes, Netherlands) and subsequent incubation for 48 h (37 \u003csup\u003eo\u003c/sup\u003eC and 5% CO\u003csub\u003e2\u003c/sub\u003e) was conducted. Afterwards, the medium was removed, and the cells were treated with the Nile Red pre-labeled NLs (NLb0, NLb1, and NLb2) dispersed in the respective cell culture medium at a final concentration of 10 \u0026micro;g/ml. After 1, 2 and 4 h incubation (37\u0026deg;C, 5% CO\u003csub\u003e2\u003c/sub\u003e), the cells were washed twice with PBS, followed by subsequent fluorescence microscopy analysis at 37\u0026deg;C (Zeiss Axio Observer Z1 inverted microscope, Zeiss, Jena, Germany), equipped with an epifluorescence illuminator and a plate heating chamber. The resulting images were processed using Carl Zeiss software (ZEN 2.6).\u003c/p\u003e \u003cp\u003eIn this direction, another set of experiments was conducted, where SH-SY5Y cells before the incubation with pre-labeled NLs with Dil (1,1'-Dioctadecyl-3,3,3',3'-Tetramethylindocarbocyanine Perchlorate) (10 \u0026micro;g/ml, 4 h, 37\u0026deg;C, 5% CO\u003csub\u003e2\u003c/sub\u003e), were incubated with pHrodo Green dextran conjugate characterized by green fluorescence in an acidic environment (for visualization of endocytic pathways). Further, the cells were stained and visualized as described above.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eb) Internalization studies of NLs in lhCMEC/D3 cell line by confocal microscopy\u003c/h2\u003e \u003cp\u003eConfocal laser scanning microscopy (Carl Zeiss, Axiovert 200M Inverted Microscope) was performed in order to confirm the internalization of NLs inside the cells and their co-localization in the cellular structures of BBB. For this purpose, cells were seeded on 35-mm glass dishes (\u0026micro;-Dish 35 mm, WillCo Glass Bottom Dishes, Netherlands) at a density of 2x10\u003csup\u003e5\u003c/sup\u003e cells per well and incubated for 24 h (37 \u003csup\u003eo\u003c/sup\u003eC and 5% CO\u003csub\u003e2\u003c/sub\u003e). After incubation (4 h, 37\u0026deg; C and 5% CO\u003csub\u003e2\u003c/sub\u003e) with the previously labeled NLs with Dil fluorescent dye, the cells were washed twice with PBS and fixed with 3.7% paraformaldehyde for 20 minutes at room temperature. After washing the cells with PBS, cytoskeleton was stained with Alexa FluorTM Phalloidin 488 green dye (6.6 \u0026micro;M) in 1% bovine serum albumin (10 minutes, 37\u003csup\u003eo\u003c/sup\u003eC), followed by nuclear staining Hoechst (hCMEC/D3), for 5 minutes at room temperature. Before microscopic visualization, Vectashield mounting medium was added to maintain and preserve the fluorescent dye. Images were processed using Carl Zeiss software (ZEN 2.6).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eStatistical analysis of the obtained results for quantitative uptake of NLs was carried out by implementing the method of least squares (PLS) using validated statistical software Simca 14.1 (Sartorius Stedim Biotech, Germany). In order to highlight the dominant independent variables that have a significant effect in the model, the VIP score was used.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results and Discussion","content":"\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eParticle size, particle size distribution and z-potential of the NLs\u003c/h2\u003e \u003cp\u003eSeveral studies have reported that despite the composition and the other surface properties (i.e. surface charge), the size range of long time circulating NPs may strongly affect their stability, \u003cem\u003ein vivo\u003c/em\u003e circulation time, as well as BBB retention and the possibility of entering into specific interactions with the BBB structures, thus their transport and uptake by the brain cells [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. In this direction, one of the initial segments was determination of the z-average diameter and z-potential of NLs in order to further correlate them to the outcome of cell uptake experiments.\u003c/p\u003e \u003cp\u003eThe z-average (hydrodynamic) diameter of the prepared NLs formulations with different amounts of PEG coated on the surface was in a range from 115\u0026ndash;130 nm with PDI\u0026thinsp;\u0026lt;\u0026thinsp;0.3 (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e) which indicates a narrow unimodal size distribution of the NLs in all formulations. According to the literature data, liposomal nano-vesicles from 100 to 140 nm exhibit longer half-life in blood circulation and avoidance of PC formation, when compared to nano-vesicles with diameter\u0026thinsp;\u0026gt;\u0026thinsp;200 nm, and also, better encapsulation efficiency than liposomes\u0026thinsp;\u0026lt;\u0026thinsp;100 nm [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. On the other hand, in the study of Nowak et al. (2019) was confirmed that spherical particles around 120 nm associate with the endothelium approximately 30-fold more than 200 nm particles, which is of extreme importance for their successful transport across BBB and consequently, efficient treatment of CNS diseases [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e].\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003ePhysical characterization of prepared NLs (n\u0026thinsp;=\u0026thinsp;6)\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"4\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eFormulation\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003ez-average\u003c/p\u003e \u003cp\u003e[d\u003csub\u003eh\u003c/sub\u003e, nm]\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003ePDI\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eZeta potential\u003c/p\u003e \u003cp\u003e[mV]\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNLb0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e127.25\u0026thinsp;\u0026plusmn;\u0026thinsp;0.12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e0.277\u0026thinsp;\u0026plusmn;\u0026thinsp;0.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e-51.96\u0026thinsp;\u0026plusmn;\u0026thinsp;2.22\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNLb1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e114.90\u0026thinsp;\u0026plusmn;\u0026thinsp;0.92\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e0.238\u0026thinsp;\u0026plusmn;\u0026thinsp;0.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e-15.77\u0026thinsp;\u0026plusmn;\u0026thinsp;0.51\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNLb2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e131.03\u0026thinsp;\u0026plusmn;\u0026thinsp;0.30\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e0.289\u0026thinsp;\u0026plusmn;\u0026thinsp;0.04\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e-37.64\u0026thinsp;\u0026plusmn;\u0026thinsp;1.42\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eTable\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e\u003c/p\u003e \u003cp\u003eFrom Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, it can be also observed a decreasing trend of the negative z-potential with the increase of PEG amount on the surface of NLs. As previously reported in our earlier study, this reverse trend is probably result of the reduced NLs electrophoretic mobility due to the hydrodynamic resistance given by PEG presence which also contributes for masking the predominant negative charge of the structural phospholipids present in the NLs (Shalabalija et al., 2021).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eStability studies of NLs in cell culture medium\u003c/h2\u003e \u003cp\u003eIn order to provide a more detailed examination of the influence of serum components present in the cell culture medium on the average NLs size, AF4-MALS/DLS analysis was performed. For this purpose, the particle size of the native formulations (NLb0, NLb1 and NLb2) was first determined, as well as after their incubation with serum-free and serum supplemented cell culture medium over 1 and 4 h. Prior performing these fractionation analyses, the z-average mean diameter of the NLs was also examined by dynamic light scattering in batch-mode, and ranged from 96.10\u0026thinsp;\u0026plusmn;\u0026thinsp;0.81 to 140.20\u0026thinsp;\u0026plusmn;\u0026thinsp;0.95 nm (PDI\u0026thinsp;\u0026lt;\u0026thinsp;0.256).\u003c/p\u003e \u003cp\u003eLiterature data suggests that Dynamic Light Scattering (DLS) is widely employed for sizing liposomes and other colloidal materials. It operates by detecting laser light scattered due to Brownian motion of particles or macromolecules in suspension, with the scattering frequency dependent on particle size, offering rapid and straightforward analyses. However, larger particles can skew results and complicate measurements in heterogeneous samples, typically requiring a five-fold difference in average size to resolve distinct populations. In contrast, Asymmetric Flow Field-Flow Fractionation (AF4) is more time-intensive but excels in fractionating samples under optimal flow and separation conditions. This method is distinguished by gentle separation conditions and a wide operational range. Unlike conventional chromatography, AF4 does not utilize a stationary phase. The separation mechanism involves a longitudinal parabolic flow profile whithin the channel.This profile induces smaller particles to elute more swiftly compared to larger particles, particularly in proximity to the semipermeable membrane [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. In particular, AF4 is a precise method for separating liposomes based on their hydrodynamic size, with particle sizes determined directly from their elution times. AF4-MALS has been extensively utilized for sizing various categories of nanoparticles such as metal oxides, polymeric and silica nanoparticles. Additionally, it has been employed for the separation of diverse macromolecules and structures including proteins, viruses and cells. These applications have facilitated the analysis of liposomes, enabling the separation of populations obtained from the same method synthesis and determination of their size. Optimizing separation variables in AF4-MALS involves several parameters such as cross-flow conditions, focusing rate and duration, sample loading, and carrier conditions. The composition of the carrier buffer, as well as its ionic strength and pH are crucial considerations for stabilizing the structures, preventing agglomeration or sedimentation, and avoiding interference with analytes and the membrane [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eFigure \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e represents the fractograms obtained from the AF4-analysis of the native formulations. The black line originates from the UV signal (230 nm) and the red line from the light scattering signal at 90\u0026deg;. Both signals are normalized to the highest signal. The UV-absorbance peaks (black signal), observed at a retention time of about 20 minutes, are most likely related to the absorption of the PEG. In addition, it can be seen that a more intense absorption peak is obtained at the same retention time for NLb1, the formulation with the highest amount of PEG on the surface, in comparison to NLb2. Moreover, it can be observed that the shape of the light-scattering (red line) and UV-signals (RT 40\u0026ndash;60 min) of those materials treated with little or no PEG (NLb0 and NLb2) looks differently compared to NLb1 (pyramid-like vs. near Gaussian like). As already mentioned in several occasions, the presence of PEG on the surface of NLs can improve the physical stability of the liposomal dispersions through steric repulsion [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. Therefore, formulations with a low amount of PEG or no PEG tend to agglomerate or lose their native structure thus leading to fragmentation. Hence, the unusual peak-shape at 20 min and the weak signal at around 60 minutes in NLb0 may be a result of the absorption of various fragments of the nanoliposomes and/or some of the components present in the soybean lecithin, as well as artefact of the initial formation of peroxides in the unsaturated fatty acid residues of the phospholipid molecules which show maximum absorbance at around 230 nm [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e].\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eFigure\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e\u003c/p\u003e \u003cp\u003eIn Figure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e, the light scattering signal at 90\u0026deg; and the related geometrical radii are overlaid for native formulations NLb0, NLb1 and NLb2. Formulation NLb1 (red lines) showed to contain slightly larger particles in the final part of the eluted peak compared to the other two formulations, probably attributable to some small, insignificant fraction of agglomerated NLs formed during the measurements. The same was confirmed by DLS in-line measurements (Fig. S2).\u003c/p\u003e \u003cp\u003eEach of the four sections (a-d) included in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e show overlaid signals obtained from the UV-detector and the z-average coming from the DLS operated in flow-mode. It can be observed that there is no significant change in the size of NLb1 vesicles (formulation with 50 mg PEG), after their incubation in cell culture medium without and with serum for a period of 1 and 4 h. This situation only confirms the stability of this formulation, and also supports the fact that PEG contributes in the prevention and suppression of the PC formation process.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eFigure\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e\u003c/p\u003e \u003cp\u003eDuring these experiments, it was also observed that formulation NLb2 incubated for 1h in the serum supplemented cell culture medium contained slightly smaller particles in the upper particle size range compared to the same sample incubated for 4 h (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eIn addition, the measurements also showed smaller NLb2 particles in the upper size range when this formulation was incubated in serum supplemented cell medium compared to when it was incubated in serum free cell medium at the both time points, separately (1 and 4 h) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea, c). In the case of particle diameter increase as a result of the PC formation, it would be expected a general increase in size throughout the whole size range to be seen. In this case, the increase was only observed in the upper size range. In this direction, the obtained results can be attributed to the fact that serum proteins can also stabilize nano-carriers, thus preventing their aggregation process (Kennedy et al., 2018).\u003c/p\u003e \u003cp\u003eFigure\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e\u003c/p\u003e \u003cp\u003eSimilar to NLb2, AF4 analysis showed that the particles of NLb0 (non-PEGylated formulation) were characterized by a smaller size when incubated in serum supplemented cell culture medium, compared to those incubated in serum free medium. As already discussed, this situation can be the result of the stabilizing effect provided by the proteins present in the serum on the NLs (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea, c). However, it is interesting to note that during these studies an unexpected decrease in the size of NLs was observed after 4 h vs. 1 h of incubation in serum supplemented medium (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eb), which is probably due to the fact that PC formation is a dynamic process that generally tends to evolve over time and involves many different driving forces controlled by the properties of nano-systems, proteins and the medium itself [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. The obtained results are in accordance to the results of the study of Miclăuş et al. (2014), where it was demonstrated that the soft corona (formed at the initial time points of incubation) contains more proteins than the hard corona formed at later time intervals, resulting in a larger particle diameter at early incubation periods [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e].\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eFigure\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003eProteomic profiling of the adsorbed proteins onto NLs surface\u003c/h2\u003e \u003cp\u003eIn the next step, qualitative analysis of the adsorbed serum components on the surface of the nano-formulations (NLb0, NLb1 and NLb2) was investigated (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). For this purpose, NLs were incubated in cell culture medium with and without serum (as control) for 1 and 4 h. From the graphical representations, it can be observed that the protein adsorption by NLb1 and NLb2 (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e) is already expressed in the first hour of incubation, resulting with strong bands at about 60 kDA, originating from albumin, the most abundant protein in the serum. On the other hand, these bands are not so expressed in NLb0. Considering that the sensitivity of the bioanalyzer is high and it covers a wide range of concentrations, the results obtained for this formulation may be due to problems with denaturation of proteins present in the formed PC, as well as the manipulation and processing of the sample. Namely, false negatives might arise because proteins detach from the nanoparticle-corona complex under the influence of centrifugal forces. Hence, it's crucial to ascertain the optimal number of washing cycles and centrifugation duration necessary for effectively isolating a particular type of nanosystem-corona complex from a protein-rich medium [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. In addition, weak bands from other proteins can be observed in all three formulations, but more detailed analysis by mass spectrometry is required.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eFigure\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003eCell uptake assessment of NLs\u003c/h2\u003e \u003cp\u003eAs previously discussed, one of the prerequisites for achieving a therapeutic effect in the brain is the successful transport of NLs across the BBB, as well as their internalization in neurons. In this direction, after determining the safety concentration range of NLS [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e], we then investigated the \u003cem\u003ein vitro\u003c/em\u003e cell uptake of NLs by two cell lines: BBB cells (hCMEC/D3) and human neuroblastoma cells (SH-SY5Y). Quantitative uptake experiments performed on the two cells lines (hCMEC/D3 and SH-SY5Y) exposed to 5, 10 or 100 \u0026micro;g/ml of the NLs under investigation and at different time-points (1, 2 and 4 h) are reported in Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e.\u003c/p\u003e \u003cp\u003eAs it can be observed, there is a gradual increment of the cell uptake for all formulations with the increase of their concentrations at all-time points for both cell lines analyzed, which is an expected phenomenon. Similar, increasing trend in the uptake can be seen with prolonged incubation time, except for the highest concentration tested (100 \u0026micro;g/ml), for which differences were found between the two cell lines. At this concentration, the uptake measured of all NLs formulations was around 3 \u0026micro;g at all incubation times for hCMEC/D3 cell line, whereas for SH-SY5Y it varied from 3.28\u0026thinsp;\u0026plusmn;\u0026thinsp;0.21 to 4.14\u0026thinsp;\u0026plusmn;\u0026thinsp;0.19 \u0026micro;g at 1 and 4 h, respectively. It is well known that the internalization of nanoliposomes into cells can take place through several energy dependent endocytic pathways (phagocytosis, clathrin-mediated endocytosis, caveolin-mediated endocytosis, clathrin/caveolae-independent endocytosis, and micropinocytosis), as well as passive transport or diffusion which is an uncompetitive movement of the nano-systems, either directly through membrane phospholipids (simple diffusion) or in combination with membrane proteins (facilitated diffusion) [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. Numerous studies have revealed that endocytosis is a process that occurs through membrane-particle adhesion followed by elastic deformation of the cell membrane and receptor diffusion to the surface of the membrane, processes highly dependent on the physico-chemical properties of the NLs, as well as their concentration and exposure time (Sabourian et al., 2020). As no significant increase of uptake was found for the highest concentration tested for all three NLs and for both cell lines, it is expected that at 1 h, hCMEC/D3 and SH-SY5Y cells have already reached their maximal endocytic potential, which is probably due to the saturation of uptake mechanisms leading to limited internalization [\u003cspan additionalcitationids=\"CR33\" citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. Additionally, as previously elaborated, the increased uptake by increasing the incubation time observed at 5 and 10 \u0026micro;g/ml for all NLs investigated confirms the lack of a saturable transport process [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e].\u003c/p\u003e\n\u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eCell uptake of NLs (\u0026micro;g) by hCMEC/D3 and SH-SY5Y cell lines\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"7\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colspan=\"4\" nameend=\"c4\" namest=\"c1\"\u003e \u003cp\u003ehCMEC/D3 cell line\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"3\" nameend=\"c7\" namest=\"c5\"\u003e \u003cp\u003eSH-SY5Y cell line\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1h\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2h\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e4h\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1h\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e2h\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e4h\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"7\" nameend=\"c7\" namest=\"c1\"\u003e \u003cp\u003e5 \u0026micro;g/ml\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNLb0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.09\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.17\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.54\u0026thinsp;\u0026plusmn;\u0026thinsp;0.09\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.19\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.28\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0.31\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNLb1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.22\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.38\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.55\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.18\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.24\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0.24\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNLb2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.20\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.23\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.29\u0026thinsp;\u0026plusmn;\u0026thinsp;0.04\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.18\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.20\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0.21\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"7\" nameend=\"c7\" namest=\"c1\"\u003e \u003cp\u003e10 \u0026micro;g/ml\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNLb0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.36\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.59\u0026thinsp;\u0026plusmn;\u0026thinsp;0.04\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1.07\u0026thinsp;\u0026plusmn;\u0026thinsp;0.12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.61\u0026thinsp;\u0026plusmn;\u0026thinsp;0.11\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.74\u0026thinsp;\u0026plusmn;\u0026thinsp;0.04\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0.85\u0026thinsp;\u0026plusmn;\u0026thinsp;0.04\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNLb1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.46\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.64\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1.12\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.61\u0026thinsp;\u0026plusmn;\u0026thinsp;0.06\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.66\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0.74\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNLb2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.51\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.56\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.70\u0026thinsp;\u0026plusmn;\u0026thinsp;0.12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.51\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.66\u0026thinsp;\u0026plusmn;\u0026thinsp;0.08\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0.72\u0026thinsp;\u0026plusmn;\u0026thinsp;0.04\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"7\" nameend=\"c7\" namest=\"c1\"\u003e \u003cp\u003e100 \u0026micro;g/ml\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNLb0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e3.20\u0026thinsp;\u0026plusmn;\u0026thinsp;0.13\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e3.38\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e3.25\u0026thinsp;\u0026plusmn;\u0026thinsp;0.06\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e3.67\u0026thinsp;\u0026plusmn;\u0026thinsp;0.10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e3.85\u0026thinsp;\u0026plusmn;\u0026thinsp;0.10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e4.14\u0026thinsp;\u0026plusmn;\u0026thinsp;0.18\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNLb1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2.92\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e3.02\u0026thinsp;\u0026plusmn;\u0026thinsp;0.07\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e3.06\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e3.28\u0026thinsp;\u0026plusmn;\u0026thinsp;0.21\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e3.27\u0026thinsp;\u0026plusmn;\u0026thinsp;0.12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e3.59\u0026thinsp;\u0026plusmn;\u0026thinsp;0.07\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNLb2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2.98\u0026thinsp;\u0026plusmn;\u0026thinsp;0.07\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e3.03\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e3.08\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e3.57\u0026thinsp;\u0026plusmn;\u0026thinsp;0.25\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e3.58\u0026thinsp;\u0026plusmn;\u0026thinsp;0.10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e3.81\u0026thinsp;\u0026plusmn;\u0026thinsp;0.11\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \n \u003cp\u003eTable\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e\u003c/p\u003e \u003cp\u003eIn order to investigate the influence of the possible independent factors (experimental conditions \u0026ndash; time of incubation/NLs concentration, type of formulation-amount of PEG) on the quantitative uptake of the NLs in the specified cell line cultures, a multivariate statistical analysis was performed. The correlation coefficients obtained from the initial model, comprised of all internalization data (normalized uptake - %) were low, but it was observed that the cell type predominantly affects the scores of the individual points, which is why it was decided PLS-DA to be performed. In continuation to the above discussed, for the internalization kinetic experiments, separate multivariate statistical models were performed for each cell culture. The internalization model in hCMEC/D3 also confirmed that the sample concentration and the exposure time were dominant factors on the percentage of NLs taken up (Fig. S3 a-c). According to the VIP plot the formulation type i.e. the amount of PEG on the surface also had a significant effect on the uptake (S3 d). The model of kinetic experiments on the SH-SY5Y cell line presents a similar behavior to the previous model, where concentration and exposure time were the dominant factors affecting uptake, while the amount of PEG on the surface has a smaller but distinctive influence (Fig S4 a-d).\u003c/p\u003e \u003cp\u003eAs it can be seen from Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e, the uptake of NLs by hCMEC/D3 after 4 h of incubation is highest for the formulation with the highest amount of PEG on the surface - NLb1 (0.55\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05 \u0026micro;g and 1.12\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02 \u0026micro;g, at 5 and 10 \u0026micro;g/ml treatment concentrations, respectively), followed by the formulation with no PEG on the surface - NLb0 (0.54\u0026thinsp;\u0026plusmn;\u0026thinsp;0.09 and 1.07\u0026thinsp;\u0026plusmn;\u0026thinsp;0.12 \u0026micro;g, at 5 and 10 \u0026micro;g/ml treatment concentrations, respectively). The lowest rate of internalization was observed in the formulation with 5 mg of PEG on the surface - NLb2 (0.29\u0026thinsp;\u0026plusmn;\u0026thinsp;0.04 and 0.70\u0026thinsp;\u0026plusmn;\u0026thinsp;0.12 \u0026micro;g, at 5 and 10 \u0026micro;g/ml treatment concentrations, respectively). Numerous research groups have demonstrated that the PC formation can significantly influence and dictate the cell recognition and the internalization, as well as the intracellular trafficking of nano-systems since it gives them new biological identity [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. Adsorbed proteins on nanostructures can hinder cell membrane adhesion and compromise stability, leading to decreased cellular uptake. The composition of formed PC on NLs affects targeting yields, nanoparticle-cell interactions, and internalization mechanisms. Nano-carriers can induce structural changes in adsorbed proteins, influencing cell signal transduction. The impact of PC on particle-cell interactions varies based on particle properties and cellular components, as well as the nature of the cell culture medium [\u003cspan additionalcitationids=\"CR38\" citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. The medium used for the cell uptake experiments of hCMEC/D3 cells was supplemented with serum as well as growth factors, dyes and antibiotics. Apart this fact, the highest cell uptake of NLb1 can be also attributed to its z-potential which is less negative (-15 mV), compared to the other two formulations, since less negative or positively charged nano-carriers would be expected to be more efficient in crossing BBB which is characterized by a negative charge [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. On the other side, several research groups have shown that the PEG surface density and conformation play a key role and improve the diffusion and transport of different types of nano-systems across endothelial barriers, particularly the BBB, and consequently, their brain distribution. Taking into consideration that NLb1 exhibits high amount of PEG, the density of the chains on the liposome surface is expected to be increased and be characterized with \u0026ldquo;dense brush\u0026rdquo; conformation as steric hindrances restrict movement and self-coiling of the grafted polymer [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]. In the research of Nance et al. (2012) it was also demonstrated that nano-systems characterized by \u0026ldquo;dense brush\u0026rdquo; PEG coating can permeate the BBB and accumulate more efficiently in the brain parenchyma ex vivo, than the uncoated [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eFrom Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e it can also be observed that during 1 h incubation intervals the cell uptake amount of NLb0 is lower compared to NLb2, at both treating concentrations (5 and 10 \u0026micro;g/ml). However, the opposite case is noticed over 4 h incubation where the quantitative cellular uptake of NLb0 was approximately 1.5 fold higher than the uncoated formulation. The main reason for this can be the fact that in our previous stability studies conducted by AF-4 analysis, it was shown that serum proteins present in the cell culture medium stabilize NLb0 in terms of preventing the process of aggregation, and additionally, unlike for NLb2, there was a decrease in the average diameter over the incubation time of 4h, which is probably due to the dynamic process of PC formation. These results only confirm the statement regarding the opposite dependence between the particle size and hCMEC/D3 liposomal uptake and adhesion as well as the alterations on the internalization promoted by the adsorbed serum proteins onto NLs surface [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eOn the other hand, NLb2, despite the low amount of PEG on its surface also showed saturable uptake within the first hour (0.20\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02 and 0.51\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02 \u0026micro;g, at 5 and 10 \u0026micro;g/ml treating concentrations, respectively), since the difference in the amount of internalized particles of NLb2 in the later incubation times (0.29\u0026thinsp;\u0026plusmn;\u0026thinsp;0.04 and 0.70\u0026thinsp;\u0026plusmn;\u0026thinsp;0.12 \u0026micro;g after 4 h, at 5 and 10 \u0026micro;g/ml treating concentrations, respectively) tended to fade out and become significantly lower compared to the other two formulations. These results additionally confirm the limited capacity of NLb2 to accumulate intracellularly and is also indicative of equilibrium between endocytosis and exocytosis [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]. Additionally, literature data suggests that PEG-coated particles with a surface charge between \u0026minus;\u0026thinsp;20 and \u0026minus;\u0026thinsp;40 mV are not capable to cross BBB probably due to the insufficient dense coating of PEG (Nance et al., 2012). This is in accordance with our results since we can conclude that hydrophilicity as well as surface charge can significantly affect the nano-system delivery to BBB, and thus brain tissues.\u003c/p\u003e \u003cp\u003eWhen it comes to the cellular uptake of the NLs by SH-SY5Y cell line, there is a different trend of quantitative internalization among the formulations. Namely, the formulation characterized by the highest cellular uptake after 4 h is the non-PEGylated NLb0 (0.31\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02 and 0.85\u0026thinsp;\u0026plusmn;\u0026thinsp;0.04 \u0026micro;g, at 5 and 10 \u0026micro;g/ml treating concentrations, respectively), followed by NLb1 (0.24\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01 and 0.74\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02, at 5 and 10 \u0026micro;g/ml treating concentrations, respectively). The same situation as in hCMEC/D3, NLb2 was observed to have the lowest cellular uptake with 0.21\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01 and 0.72\u0026thinsp;\u0026plusmn;\u0026thinsp;0.04 \u0026micro;g, at 5 and 10 \u0026micro;g/ml treating concentrations, respectively. Obtained results are in accordance with the literature data suggesting that non-PEGylated liposomes are prone to more efficient uptake by neuroblastoma cells. This outcome is probably due to the fact that PEG chains hinder the interactions of the liposomes with different membrane structures of this type of cells, thus resulting with poor intracellular transport [\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e]. On the other hand, neurons exhibit membranes which are unique in its composition being highly enriched in lipids, in particular cholesterol, which plays key role in regulation of the membrane structure, fluidity and permeability as well as multiple aspects of the synaptic transmission [\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e]. In pure human SH-SY5Y cell cultures, the glia-derived cholesterol is non-existing, and addition of cholesterol is needed in order to achieve conditions resembling normal neuronal environment with surrounding glial cells, as well as to promote the process of the SH-SY5Y neuroblastoma cell differentiation into neuronal cell type [\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e]. Up to date, several findings reported the clear preference of SH-SY5Y neurons for cholesterol containing liposomes. Namely, Lee et al. (2013) reported that the addition of cholesterol into the liposomal formulation resulted with 11-fold enhanced uptake by this cell culture line, implying on the fact that the composition of NLs significantly affects their uptake by neuronal cells and are avidly taken up by the addition of cholesterol [\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e]. In this sense, since all three nano-formulations contain cholesterol into their lipid bilayer, the lower uptake of the PEGylated liposomes (NLb1 and NLb2) may be a result of the steric effect of the PEG chains onto the surface, which probably act as a barrier and prevent the access of cholesterol to the cellular structures. It is also important to be mentioned that PEGylation can prevent or reduce, but does not totally exclude the protein binding to the NLs surface. Excessive PEGylation may contribute to less efficient binding with protein targets that would work as ligands for receptor mediated transport and delivery, finally resulting with partial inhibition and reduction of the cellular uptake (Pozzi et al., 2014).\u003c/p\u003e \u003cp\u003eTaken into consideration all above mentioned, it can be summarized that cellular transport and internalization are influenced by numerous features such as the NLs composition (especially the amount of PEG), physico-chemical properties of NLs, the experimental conditions (concentration and incubation time), as well as the composition of the cell medium and the structural characteristics of cell culture lines.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003eCell uptake assessment of NLs in presence of transport pathways inhibitors\u003c/h2\u003e \u003cp\u003eIn order to have insights into the mechanism of internalization of the NLs, as well as to better understand and correlate with the previously presented quantitative results for cell internalization at 37\u0026deg;C, uptake experiments in presence of specific inhibitors of endocytotic pathways were performed. In this sense, the cell culture lines (hCMEC/D3 and SH-SY5Y) were pretreated (40 minutes) with chlorpromazine or indomethacin as specific inhibitors of chlatrin and caveolin-mediated endocytosis. In addition, uptake experiments at 4\u0026deg;C were conducted, when it is supposed that all ATP-dependent transport mechanisms are blocked (Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). The fluorescence of cells incubated with NLs at 37\u0026deg;C was considered as 100%, while the fluorescence after incubation in the presence of inhibitors was expressed as a relative percentage compared to the cells without inhibitor.\u003c/p\u003e \u003cp\u003eThe statistical analysis of the obtained results for hCMEC/D3 cells (Fig. S5 a-c) clearly shows the concentration and endocytosis inhibitors having a significant effect, while lowering the temperature of the experiment (total energy metabolism) had no significant effect on the total uptake. In addition, the type of formulation, i.e. the amount of PEG on the NLs surface also affects the uptake, as seen on the VIP plot which provides an overall representation of the effect of the independent variables (Fig. S5 d). The SH-SY5Y uptake pattern showed a different trend of the influence of the independent variables (Fig. S6 a-c). According to the VIP plot (Fig. S6 d), it can be concluded that the concentration of the sample and also the temperature of the experiment are dominant factors affecting the uptake. Similarly, as with hCMEC/D3, type of formulation or more precisely, PEG amount also demonstrated a significant effect on the uptake under varying experimental conditions.\u003c/p\u003e \u003cp\u003eFrom Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e can be observed that incubation at 4\u0026deg;C induces cell metabolic inhibition, resulting (for the concentration of 10 \u0026micro;g/ml) in a reduction of ~\u0026thinsp;30% of the uptake of all formulations in both cell lines, compared to the experiments performed at 37\u0026deg;C. This indicates that energy-dependent endocytosis is included in the NLs uptake along with physical adhesion or passive diffusion [\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e].\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eFigure\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e\u003c/p\u003e \u003cp\u003eIn order to investigate the mechanism of endocytosis, cells were also treated with chlorpromazine which is known to inhibit AP2, one of the key adaptor proteins in clathrin-mediated endocytosis and it is also involved in clathrin accumulation in late endosomes, thereby inhibiting coated pit endocytosis. Figure\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e shows a decrease in the uptake of NLb1 and NLb2 by \u0026sim;25% in both cell lines compared to the control at 37\u0026deg;C, referring to the fact that chlatrin-mediated endocytosis may be involved, one of the predominant internalization pathways for the uptake of NPs \u0026sim;120 nm, because of the size of clathrin coated pits [\u003cspan additionalcitationids=\"CR50\" citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e]. Additionally, the performed experiments resulted with significant reduction in the uptake of NLb0 in hCMEC/D3 (50.59\u0026thinsp;\u0026plusmn;\u0026thinsp;2.65%), whereas only a slight decrease was observed in SH-SY5Y (93.78\u0026thinsp;\u0026plusmn;\u0026thinsp;4.58%). This could be due to the different structural specificities and the distinct cell surface properties as well as the specific PC formed onto the surface of NLs after incubation with the cell culture medium [\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e]. On the other hand, it should be taken into consideration that when attempting to block a certain transport pathway, different types of cells usually adapt \u003cem\u003evia\u003c/em\u003e activation of alternative mechanisms as well as overcompensation for the blocked function or receptor (Francia et al., 2019). This statement can further explain the heterogeneous results obtained for the inhibition of caveolin-mediated endocytosis with indomethacin between the different formulations in the different cell lines (49.96\u0026thinsp;\u0026plusmn;\u0026thinsp;2.95\u0026ndash;87.10\u0026thinsp;\u0026plusmn;\u0026thinsp;3.56% and 57.17\u0026thinsp;\u0026plusmn;\u0026thinsp;1.56\u0026ndash;92.38\u0026thinsp;\u0026plusmn;\u0026thinsp;2.65%, for hCMEC/D3 and SH-SY5Y, respectively). The surface properties of the nano-systems such as PEGylation can also affect the cell uptake/adhesion since PEG chains conformation and also, the aggregation of PEG polymers in the contact region between a PEGylated liposome and the membrane can influence the membrane wrapping process of PEGylated liposomes during endocytosis [\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e]. Therefore, it can be that different energy-dependent and non-dependent pathways are probably included in the dictation of NLs transport across BBB and neurons.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec21\" class=\"Section2\"\u003e \u003ch2\u003eCell uptake experiments on co-cultured hCMEC/D3 and SH-SY5Y cell line\u003c/h2\u003e \u003cp\u003eSeveral studies have reported the internalization and uptake of NLs by different types of neuronal and BBB cell culture lines, individually. In this sense, detailed experiments were conducted under different experimental conditions on the two cell culture lines, hCMEC/D3 and SH-SY5Y, in order to determine the quantitative cell uptake and predict the internalization mechanism of the NLs investigated. However, despite the confirmed uptake of SH-SY5Y neuroblastoma cells after direct exposure to NLs presented earlier, it is not certain whether the results would be consistent and the obtained effects would be replicated \u003cem\u003ein vivo\u003c/em\u003e, where the ability of nano-carriers to serve as platforms for active components intended for CNS treatment is limited due to the primary challenge of permeating across the BBB [\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e]. Another thing that should be taken into consideration is the fact that the information regarding the fate of the nano-systems in pericytes, astrocytes or neurons after having crossed the BBB is quite limited [\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e]. For this reason, the human derived brain endothelial cells, hCMEC/D3, were cultured on the apical side of permeable Transwell inserts, while monolayers of SH-SY5Y neuroblastoma cells were seeded on the basal side of 12-well plates chambers. After hCMEC/D3 reached a TEER value\u0026thinsp;\u0026gt;\u0026thinsp;230 Ω and the confluence of SH-SY5Y was \u0026gt;\u0026thinsp;85%, both cell culture lines were combined and transport studies of the three NLs formulations were performed.\u003c/p\u003e \u003cp\u003eThe results from the NLs\u0026rsquo; cellular uptake into neuronal cells after crossing the blood-brain barrier \u003cem\u003ein vitro\u003c/em\u003e in our study suggest that the non-PEGylated formulation (NLb0) is internalized in highest percent (27.54\u0026thinsp;\u0026plusmn;\u0026thinsp;2.93%), followed by NLb2 (26.46\u0026thinsp;\u0026plusmn;\u0026thinsp;1.87%) and by the formulation with the highest amount of PEG onto its surface - NLb1 (25.17\u0026thinsp;\u0026plusmn;\u0026thinsp;1.74%). This implies the successful transport of these liposomal nano-carriers across a BBB model and the consequent uptake of the particles by the neuronal cells [\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e]. It was also noticed that the quantitative uptake trend by SH-SY5Y in combination with hCMEC/D3 for the three different formulations is in good relation to the experiments on the single cell line (33.27\u0026thinsp;\u0026plusmn;\u0026thinsp;1.95, 25.17\u0026thinsp;\u0026plusmn;\u0026thinsp;2.65 and 26.46\u0026thinsp;\u0026plusmn;\u0026thinsp;1.54% for NLb0, NLb1 and NLb2, respectively), which could be attributed to the physico-chemical properties of NLs and physiological factors affecting their internalization as well as the morphological properties of SH\u0026mdash;SY5Y already discussed.\u003c/p\u003e \u003cp\u003eRecent studies have highlighted the pivotal role of co-culture models in advancing \u003cem\u003ein vitro\u003c/em\u003e neurotoxicity research. These models have significantly contributed to bridging the gap in faithfully replicating the human BBB phenotype. This fidelity is crucial for conducting permeability studies, assessing neurotoxicity, and investigating aspects related to neurodegenerative diseases [\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e]. The findings from the study of Freese et al. (2014) illustrates the effectiveness of a new hCMCEC/D3 \u0026ndash; SH-SY5Y bio-assay \u003cem\u003ein vitro\u003c/em\u003e system which proves adept at predicting drug penetration across the BBB, particularly for drugs relevant to Alzheimer's disease (AD) therapy [\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e]. Тhis same \u003cem\u003ein vitro\u003c/em\u003e model with slight modifications was used by Mursaleen et al. (2021) in order to demonstrate that micellar nanocarriers loaded with hydroxytyrosol effectively crossed the BBB \u003cem\u003ein vitro\u003c/em\u003e without inducing cytotoxicity. Moreover, these nanocarriers protected neuronal SH-SY5Y cells against rotenone-induced oxidative stress, as assessed by mitochondrial hydroxyl levels [\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e]. However, it is important to note that in this study, the evaluation of results was done based on the biological activity of the drug, not through measurement of the micelle carriers permeation and uptake by brain cells.\u003c/p\u003e \u003cp\u003eWhen it comes to permeability studies of lipid nanoparticles with different surface characteristics across hCMEC/D3 and their subsequent uptake in SH-SY5Y, the literature is limited and generally focused on research involving ligand-functionalized lipid nano-systems. In this context, one of the few studies available is evaluation of the efficacy of apolipoprotein E (APOE) targeting nanoparticles for delivering donepezil across the BBB. The results underscored the effective permeability of targeting nanoparticles across the BBB and the findings indicated that nanoparticles equipped with APOE targeting ligand demonstrated higher cellular uptake compared to the non-functionalized ones [\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec22\" class=\"Section2\"\u003e \u003ch2\u003eInternalization studies\u003c/h2\u003e \u003cp\u003eTo further verify the abovementioned results, the internalization of NLs by hCMEC/D3 and SH-SY5Y cell lines was investigated using fluorescent live-cell imaging and confocal microscopy. Figure\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e (a-c) and 8 (a-c) show images obtained by fluorescent microscopy of NLs incubated in both cell lines for 1, 2 and 4 h. From the microscopic images, the time-dependent internalization of all three formulations can be observed in both cell lines, where higher amounts of internalized nanoliposomal vesicles were noted in later time intervals. Additionally, from the fluorescence intensity, it can be seen that in the cells of the blood-brain barrier, the largest amount of internalized vesicles is attributed to NLb1, followed by NLb0 and NLb2, respectively, whereas in neuroblastoma cells, NLb0 exhibits the highest percentage of uptake, which is consistent with previous studies on the quantitative uptake at 37\u0026deg;C.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eFigure\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e\u003c/p\u003e \u003cp\u003eFigure\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003e\u003c/p\u003e \u003cp\u003eMany research studies have demonstrated that liposomes tend to follow an endocytic mechanism of cellular internalization. Therefore, it was also important to visualize the NLs internalization pathway and confirm their co-localization in endosomes. In this direction, NLs were incubated for 4 h in the presence of a dye that signals endocytosis, i.e. in an acidic environment (endosomes and lysosomes) gives green fluorescence. From the presented images on Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003e (a-c) it could be seen and confirmed that all formulations of NLs have been internalized and co-localized in the endosomal compartments in SH-SY5Y cells.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eFigure\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003e\u003c/p\u003e \u003cp\u003eFrom the images obtained by confocal microscopy, the internalization of NLs can be confirmed in hCMEC/D3 cell line. Literature data suggests on the ability of lipid NLs to cross BBB despite its highly restrictive nature, and moreover, deliver the encapsulated drugs in different cell compartments [\u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e]. When it comes to nanoliposomes, as it could be seen from the presented images on Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e10\u003c/span\u003e (a-c), they show tendency of accumulation around the perinuclear area. [\u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e]. Regarding the intracellular localization of NLs, the obtained results showed that there is no difference in the cell distribution of the different NLs formulations, or more precisely, the presence and the amount of PEG on their surface did not influence the intracellular NLs co-localization (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e10\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eFigure\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e10\u003c/span\u003e\u003c/p\u003e \u003c/div\u003e"},{"header":"Conclusion","content":"\u003cp\u003eIn this work three different nanoliposomal formulations with different PEG amounts on the surface were prepared and appropriately characterized in a biorelevant manner. The results from the stability studies of the tested formulations confirmed that after incubation in cell culture medium there were no changes in the mean z-size of the sample with the highest amount of PEG (NLb1) which also confirmed the stability of this formulation. Additionally, serum proteins were found to likely stabilize the PEG-free formulation (NLb0) in terms of preventing the aggregation process. Furthermore, by electrophoresis experiments, it was evident that protein corona was formed within the first hour of incubation in the serum supplemented culture medium, and the protein that was adsorbed in the largest percentage on the surface of NLs was albumin. Statistical analysis performed on the cell uptake pattern showed that NLs concentration and incubation time play a key role on the percentage of internalized NLs. Furthermore, the highest uptake by hCMEC/D3 cell line was obtained for the formulation with the highest amount of PEG on the surface (NLb1). A different situation was observed for the cellular uptake by SH-SY5Y, where the PEG-free formulation (NLb0) gave the most successful internalization. When it comes to the mechanism of cellular internalization, all nano-vesicle samples were characterized by energy-dependent endocytic transport and passive diffusion. The transport studies on the combined hCMEC/D3/SH-SY5Y cell line confirmed the successful transport of the nanoformulations across the BBB and their subsequent uptake by the neuroblastoma cells. The obtained micrographs from the fluorescent microscopy on live cells and the confocal microscopy gave insight into the successful internalization of the NLs in the BBB and neuroblastoma cells, in addition revealing that the co-localization of the NLs was in the perinuclear cell regions. From the above-mentioned, it can be concluded that all properties and performances of the designed NLs are in favor of the efficient brain delivery, and hence their potential for treatment of different CNS diseases.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cem\u003eEthics approval and Consent to participate\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eThis is an \u003cem\u003ein vitro\u003c/em\u003e study and no human participants were involved, neither their data or biological material.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eConsent for publication\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eIn this study no human participants were involved, neither their data or biological material. Therefore, consent for publication is not available.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eCompeting interests\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eFunding\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eExperimental data were in part generated under the Framework for Access to the Joint Research Centre Physical Research Infrastructures of the European Commission (Project: Proteomic profiling of the protein corona formed onto nanoparticles\u0026rsquo; surface upon their exposure in HCMEC/D3 cell culture medium (PPPCNCCM), Research Infrastructure Access Agreement N\u0026deg; 36025/9; \u0026nbsp;Call 2020-1-RD-Nanobiotech) and at the Department of Pharmaceutical Technology and Biopharmacy, Institute of Pharmaceutical Sciences at the University of Graz, Austria, as part of the CEEPUS student mobility program, through the CEKA PharmTech network (CIII-RS-1113-02-1819-Central European Knowledge Alliance for Teaching, Learning and Research in Pharmaceutical Technology).\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eAuthors\u0026rsquo; contributions\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eAll authors contributed to the study conception and design. Investigation, experimental work, data analysis and interpretation were performed by Dushko Shalabalija and Ljubica Mihailova. The first draft of the manuscript was written by Dushko Shalabalija and Ljubica Mihailova and all authors commented on previous versions of the manuscript. Nikola Geskovski, Otmar Geiss, Sabrina Gioria and Diletta Scaccabarozzi were actively involved in the investigation, data analysis as well as writing and reviewing the manuscript. Andreas Zimmer and Marija Glavas Dodov besides the study conception and design, they also contributed with data analysis, reviewing and editing as well as supervision of the research activities. All authors read and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eData Availability Statement\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eMistretta M, Farini A, Torrente Y, Villa C. Multifaceted nanoparticles: emerging mechanisms and therapies in neurodegenerative diseases. Brain J Neurol. 2023;146(6):2227\u0026ndash;40. \u003c/li\u003e\n\u003cli\u003eBarar J, Rafi MA, Pourseif MM, Omidi Y. Blood-brain barrier transport machineries and targeted therapy of brain diseases. BioImpacts BI. 2016;6(4):225\u0026ndash;48. \u003c/li\u003e\n\u003cli\u003eHernandez C, Shukla S. Liposome based drug delivery as a potential treatment option for Alzheimer\u0026rsquo;s disease. 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J Drug Deliv. 2011;2011:296151. \u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"nanoliposomes, surface characteristics, stability, cell uptake, internalization, cell co-culture","lastPublishedDoi":"10.21203/rs.3.rs-4828653/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4828653/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eIn contemporary research, there is a clear emphasis on the physicochemical characteristics and effectiveness of nanoliposomal (NLs) formulations. However, there has been minimal focus on elucidating nano-bio interactions and understanding the behavior of these formulations at organ and cellular levels. Specifically, it is widely recognized that when exposed to biological fluids, nano-delivery systems, including NLs, rapidly interact with various biomolecules which have a significant impact on the functionality and destiny of the nano-systems but also influence cellular biological functions. Hence, the primary objective of this study was to illuminate the evolution of physicochemical characteristics and surface properties of NLs in biorelevant media. Additionally, in order to point out the influence of specific characteristics on the brain targeting potential of these formulations, we investigated NLs interactions with BBB (hCMEC/D3) and neuroblastoma cells (SH-SY5Y) under different conditions. The results obtained from \u003cem\u003ein vitro\u003c/em\u003e comparative cell uptake studies on both cell culture lines after treatment with 3 different concentrations of fluorescently labelled NLs (5, 10 and 100 μg/mL) over a period of 1, 2 and 4 h showed a time- and concentration-dependent internalization pattern, with high impact of the surface characteristics of the different formulations. In addition, transport studies on hCMEC/D3/SH-SY5Y co-culture confirmed the successful transport of NLs across the BBB cells and their subsequent uptake by neurons (ranging from 25.17 to 27.54%). Fluorescence and confocal microscopy micrographs revealed that, once internalized, NLs were concentrated in the perinuclear cell regions.\u003c/p\u003e","manuscriptTitle":"Influence of surface characteristics on the in vitro stability and cell uptake of nanoliposomes for brain targeting","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-09-06 16:49:40","doi":"10.21203/rs.3.rs-4828653/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"17b1210f-a5b8-442a-8b8e-aa41486763c0","owner":[],"postedDate":"September 6th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-01-19T18:06:30+00:00","versionOfRecord":[],"versionCreatedAt":"2024-09-06 16:49:40","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4828653","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4828653","identity":"rs-4828653","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}