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Research Article
In vitro evaluation of antioxidant and neuroprotective effects of berberine loaded in nanostructured lipid carriers
expand article infoRadostina Bogdanova‡, Magdalena Kondeva-Burdina‡, Teodora Popova‡, Borislav Tzankov‡, Christina Voycheva‡, Marta Slavkova‡, Virginia Tzankova‡, Denitsa Stefanova‡
‡ Medical University of Sofia, Sofia, Bulgaria

Corresponding author: Denitsa Stefanova ( denitsa.stefanova@pharmfac.mu-sofia.bg )

Academic editor: Denitsa Momekova
© 2025 Radostina Bogdanova, Magdalena Kondeva-Burdina, Teodora Popova, Borislav Tzankov, Christina Voycheva, Marta Slavkova, Virginia Tzankova, Denitsa Stefanova.
This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Citation: Bogdanova R, Kondeva-Burdina M, Popova T, Tzankov B, Voycheva C, Slavkova M, Tzankova V, Stefanova D (2025) In vitro evaluation of antioxidant and neuroprotective effects of berberine loaded in nanostructured lipid carriers. Pharmacia 72: 1-14. https://doi.org/10.3897/pharmacia.72.e153730
Open Access

Abstract

Berberine (BRB) is a natural alkaloid with diverse pharmacological properties, including antitumor, anti-inflammatory, and glucose-lowering effects. Recently, its neuroprotective potential has garnered attention due to its role in mitigating oxidative stress, protein aggregation, and inflammation – key factors in neurodegenerative diseases. However, BRB’s therapeutic application is limited by poor solubility, low bioavailability, and extensive metabolism. To address these challenges, nanotechnology-based drug delivery systems have been explored. Among them, nanostructured lipid carriers (NLCs) offer an attractive strategy due to their biocompatibility, high drug loading capacity, and ability to enhance BRB’s solubility and stability. Additionally, intranasal administration of NLCs could facilitate direct nose-to-brain delivery, bypassing systemic metabolism and reducing potential side effects. The aim of this study was to develop and characterize a nanostructured lipid carrier formulation incorporating BRB (NLC-B) to enhance its solubility and neuroprotective effects. The physicochemical stability of the NLC-B system was assessed, and its ability to retain BRB’s antioxidant and neuroprotective properties was evaluated using various in vitro models. These included a 6-hydroxydopamine-induced neurotoxicity model in isolated rat brain synaptosomes, a tert-butyl hydroperoxide-induced oxidative stress model in isolated rat brain mitochondria, and an iron/ascorbate-induced lipid peroxidation assay in isolated rat brain and liver microsomes. The findings from this study provide insights into the feasibility of NLC-B as a promising drug delivery system for enhancing the therapeutic potential of berberine in neurodegenerative disease treatment.

Keywords

neuroprotection, in vitro, subcellular structures, berberine, nanostructured lipid carriers

Introduction

Berberine (BRB) is a natural benzylisoquinoline alkaloid with a long history of use in traditional medicine, dating back thousands of years. It has been extensively studied for its therapeutic potential and has demonstrated numerous health benefits. Both BRB and its synthetic derivatives have been investigated for their applications in the treatment of various diseases, based on its pharmacological properties such as antitumor activity (Almatroodi et al. 2022), a significant potential in managing diabetes (Xie et al. 2022), weight loss in obesity (Ilyas et al. 2020), anti-inflammatory properties (Mohammadian Haftcheshmeh and Momtazi‐Borojeni 2021) and inhibition of carotid atherosclerosis by modulating the PI3K/AKT/mTOR signalling pathway, suggesting its potential in preventing and managing atherosclerosis (Song and Chen 2021).

The use of berberine in the treatment of neurodegenerative diseases has gained significant attention in recent years. Oxidative stress, protein aggregation, and inflammation are common pathological features of these disorders (Helman and Murphy 2016). The overproduction of reactive oxygen species (ROS), including H₂O₂, O₂•, •OH, RO•, and ROO•, plays a key role in the pathogenesis of neurodegenerative diseases. These molecules are primarily generated in mitochondria during aerobic metabolism or enter cells due to infections and environmental pollutants. Under normal conditions, cells regulate ROS levels through various enzymatic systems, but stress can impair this ability. BRB exerts its neuroprotective effects through various mechanisms, including antioxidant properties, modulation of enzyme activity, regulation of neurotransmission, and interaction with molecular targets (Tian et al. 2023). However, the most prominent and important mechanism is its antioxidant activity. Studies measuring free radical levels have demonstrated that berberine, at concentrations ranging from 50 μM to 1 mM, reduces ROS in a concentration-dependent manner (Xu et al. 2017).

A possible mechanism of BRB’s neuroprotective effect is its ability to reduce superoxide dismutase and choline acetyltransferase enzyme levels, as observed in neurodegenerative animal models. BRB has also been shown to solubilize β-amyloid plaques in mouse models of Alzheimer’s disease (Velmurugan et al. 2018).

In Parkinson’s disease models, it prevents dopaminergic neuron death by promoting autophagy and suppressing inflammation through inhibition of the NLRP3 (Huang et al. 2021). Autophagy is essential for removing damaged organelles and misfolded proteins, contributing to neuronal health.

However, berberine’s practical application is hindered by its poor solubility, permeability, extensive metabolism together with being a substrate for the P-glycoprotein efflux system (Thomas et al. 2021). Even though berberine can pass the blood-brain barrier, its pharmacokinetic features render its limited absorption (bioavailability less than 1%) (Tan et al. 2013). This would require the administration of higher doses in order to exert its neuroprotective effects. Nanotechnology is immensely studied in order to solve the mentioned issues for different drugs. The loading of active constituents in different nanocarriers can enhance the solubility, modify the drug release and affect the biodistribution (Chien et al. 2023). Interesting drug delivery systems are the lipid-based nano formulations due to their biocompatibility, biodegradability and versatility. The lipids used are considered generally regarded as safe and it is expected that such carriers would be well tolerated by the human body (Costa et al. 2021a). Among the lipid-based nanoparticles, solid lipid nanocarriers (SLNs), and nanostructured lipid carriers (NLCs) appear additionally attractive due to their industrial scalability and increased drug payload for both hydrophilic and lipophilic substances (Viegas et al. 2023). The NLCs, which are second-generation lipid-based nanoparticles, consist of solid and liquid physiological lipids and are stabilized with suitable surfactants. They are superior to SLNs in terms of higher drug loading capacity and decreased drug expulsion during storage (Elmowafy and Al-Sanea 2021). Additionally, in comparison to nano emulsions they allow the utilization of smaller surfactant concentrations, which makes NLCs better tolerated via different administration routes (Cunha et al. 2021). In terms of improving the neuroprotective effects, NLCs administered by the intranasal route could offer additional benefits. The lipid carrier can protect the payload from enzymatic degradation (Schwarz and Merkel 2019) and reduce the systemic exposure to the drug. This is a prerequisite for ameliorating adverse effects and potential increase in the therapeutic potential (Djupesland et al. 2014). Furthermore, there is data suggesting their superiority in comparison to polymer nanocarriers due to the ability to adhere to the olfactory epithelium and allowance of direct absorption through the trigeminal and olfactory nerves (Costa et al. 2021a). This makes them an attractive drug delivery system for nose-to-brain delivery.

The aim of the current study was to investigate the feasibility of incorporating BRB into nanostructured lipid carriers (NLC-B) to improve berberine’s solubility and offer potential intranasal delivery. The physicochemical stability of the proposed system was also investigated. Furthermore, we evaluated the ability of NLC-B to retain the antioxidant and neuroprotective properties of BRB, using various in vitro models, such as a model of 6-hydroxydopamine-induced neurotoxicity in isolated rat brain synaptosomes, a tert-butyl hydroperoxide-induced oxidative stress model in isolated rat brain mitochondria and a model of iron/ascorbate-induced lipid peroxidation to assess the antioxidant protection in isolated rat brain and liver microsomes.

Materials and methods

Materials

Berberine hydrochloride (BRB) was obtained from Fluorochem (Hadfield, UK). As a liquid lipid Mygliol® 812N (caprylic/capric triglyceride) was kindly gifted by IOI Oleo GmbH, Hamburg, Germany. Precirol® 5 ATO was selected as a solid lipid and its sample was donated by Gattefosse (Saint-Priest, France). Tween 20 was sourced by Sigma Aldrich (Merck KGaA, Darmstadt, Germany). All other materials used were of analytical grade. Distilled water was prepared in the laboratory. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), 6-hydroxidopamine (6-OHDA), tert-butyl hydroperoxide (t-BuOOH), Percoll, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), sucrose, 2-thiobarbituric acid (4,6-dihydroxypyrimidine-2-thiol) and Folin reagent were obtained from Sigma Aldrich, Germany; NaCl, KCl, CaCl2.2H2O, MgCl2, NaHPO4, D-glucose, trichloroacetic acid, 2,2’-dinitro-5,5’- dithiodibenzoic acid (DTNB), sulfuric acid, n-butanol, FeSO4 and Ascorbic acid were obtained from Merck, Germany.

Methods

Nanoparticles preparation and characterization

Nanostructured lipid carriers with (NLC-B) and without berberine (NLC) were prepared according to the previously described procedure by hot-emulsification and ultrasonication technique (Stefanova et al. 2025). Their stability was evaluated over 6 months of storage at room temperature in the dark.

The encapsulation efficiency (EE, %) and loading capacity (LC, %) of the NLC-B were investigated upon preparation and after storage based on the total amount of BRB added and the free BRB isolated upon centrifugation (15 100 × g) and filtration (0.45 µm) following the equations:

EE(%)= total BRB- free BRB total BRB×100

LC(%)= total BRB- free BRBNCL-B×100

The free BRB amount was determined by UV-spectrophotometer (Thermo Scientific Nicolet, Waltham, Massachusetts, U.S.) at 420 nm.

The NLC and NLC-B were characterized in terms of size, polydispersity index (PDI) and zeta potential immediately after preparation and upon 6 months of storage. The measurements were conducted in triplicate by photon-correlation spectroscopy with the help of Zetamaster (Malvern Instruments, Worcestershire, UK). The samples were prepared by 100-fold dilution with distilled water and measured at 25 °C and scattering angle of 90°. One-way ANOVA was applied to evaluate the statistical significance of the results with significance level p < 0.05.

The potential intranasal applicability was evaluated by the effect of simulated nasal fluid (SNF) on the nanoparticle’s physicochemical stability. The hydrodynamic diameter, PDI and zeta potential were evaluated by dilution of the samples with freshly prepared SNF instead of distilled water. The SNF contained 7.45 g/L NaCl, 1.29 g/L KCl, and 0.32 g/L CaCl2.2H2O and the pH was adjusted to 6.4 with triethanolamine (Trenkel and Scherließ 2021).

The in vitro release study was carried out by the dialysis bag method in 100.0 ml SNF at a temperature of 37 ± 0.5 °C and 100 rpm horizontal agitation. The regenerated cellulose dialysis membrane (12–14 kDa, Spectra/Por®, VWR International GmbH, Darmstadt, Germany) was soaked overnight in the SNF. Afterwards, a sample containing approximately 3 mg free berberine aqueous dispersion or NLC-B was placed and carefully clamped in the dialysis bag. The BRB amount was selected based on other experiments for suitable comparison purposes (Abo El-Enin et al. 2022; Mazahir et al. 2025) and to achieve sink conditions in the release medium. At predetermined time intervals aliquot samples were withdrawn and replenished with fresh medium. The cumulative amount of released BRB was determined spectrophotometrically as described earlier.

Animals

Three animals were used in the experiments. The animals were obtained from the National Breeding Centre of the Bulgarian Academy of Sciences, Sofia, Bulgaria, and kept under standard conditions in plexiglass cages with free access to water and food and 12 h/12 h light/dark regime at 20°–25 °C. Twelve hours before each specific study, the animals’ food was withdrawn. The experiments were conducted by the Ordinance No. 15 on Minimum Requirements for the Protection and Welfare of Experimental Animals (SG No. 17, 2006) and the European Regulation for the Handling of Experimental Animals. The study was approved by the Bulgarian Food Safety Agency, with permission No. 273, valid until 20.07.2025.

Isolation of sub-cellular fractions

Rat brain synaptosomes and mitochondria

Synaptosomes and brain mitochondria were isolated using a differential centrifugation by using differential centrifugation with a Percoll gradient (Taupin et al. 1994; Sims and Anderson 2008). The process utilized two buffers: Buffer A (5 mM HEPES, 0.32 M sucrose) for homogenization and Buffer B (290 mM NaCl, 0.95 mM MgCl2×2H2O, 10 mM KCl, 2.4 mM CaCl2×2H2O, 2.1 mM NaH2PO4, 44 mM HEPES, and 13 mM D-glucose) for subsequent steps. The brain homogenate prepared in Buffer A was centrifuged twice at 1,000 × g for 10 minutes at 4 °C, and the pooled supernatants underwent three additional centrifugation steps at 10,000 × g for 20 minutes at 4 °C. Synaptosomes and mitochondria were further separated using a Percoll gradient, prepared from a 90% stock solution diluted to create 16%, 10%, and 7.5% solutions. Four millilitres each of 16% and 10% Percoll were layered, followed by 7.5% Percoll containing the final pellet. The tubes were centrifuged at 15,000 × g for 20 minutes at 4 °C, resulting in three distinct layers: the bottom layer containing mitochondria, the middle layer (between the 16% and 10% Percoll) containing synaptosomes, and the top layer containing lipids. The synaptosomal and mitochondrial layers were carefully extracted, washed with Buffer B, and centrifuged again at 10,000 × g for 20 minutes at 4 °C. Finally, the isolated synaptosomes and mitochondria were diluted in Buffer B to a protein concentration of 0.1 mg/mL, and the protein content was quantified. This protocol ensured the efficient isolation of pure synaptosomal and mitochondrial fractions for further analysis.

Rat brain microsomes

The brain tissue was homogenized in a 9:1 ratio with 0.1 M Tris buffer, containing 0.1 mM dithiothreitol, 0.1 mM phenylmethylsulphonyl fluoride, 0.2 mM EDTA, 1.15% KCl, and 20% (v/v) glycerol, adjusted to pH 7.4. The homogenate was subjected to centrifugation twice at 17,000 × g for 30 minutes. The supernatants from both centrifugations were pooled and further centrifuged twice at 100,000 × g for 1 hour. The resulting pellet was stored in the Tris buffer at −20 °C until further use (Ravindranath and Anandatheerthavarada 1990).

Rat liver microsomes

Livers were perfused with 1.15% KCl and homogenized in four volumes of ice-cold 0.1 M potassium phosphate buffer (pH 7.4). The resulting homogenate was centrifuged at 9,000 × g for 30 minutes at 4 °C to isolate the post-mitochondrial fraction (S9). This fraction was then subjected to further centrifugation at 105,000 × g for 60 minutes at 4 °C. The microsomal pellets obtained were re-suspended in 0.1 M potassium phosphate buffer (pH 7.4) containing 20% glycerol. Aliquots of liver microsomes were stored at −70 °C until analysis (Simeonova et al. 2010). The protein content of the microsomes was quantified using the method of Lowry et al. (1951).

Treatments with NLC, free BRB and NLC-B

Immediately after harvesting, the isolated rat brain synaptosomes, microsomes, and mitochondria were incubated with the nanostructured lipid carriers (NLC) (10, 50, 100, 250 and 500 µg/ml), berberinе (BRB) and berberinе loaded in nanostructured lipid carriers (NLC-B) (berberinе concentrations 1, 5, 10, 25 and 50 µM) for 1 h.

Protein concentrations in all sub-cellular fractions were determined using the method of Lowry et al (1951).

In vitro neurotoxicity and lipid peroxidations models

Model of 6-OHDA-induced neurotoxicity on isolated synaptosomes

Synaptosomes were exposed to 150 μM of 6-hydroxydopamine (6-OHDA) for a duration of 1 hour, providing a controlled environment to study the mechanisms underlying Parkinson’s Disease (PD)-related neuronal damage. Following the incubation with the test compounds and 6-hydroxydopamine (6-OHDA), synaptosomes were subjected to three centrifugation cycles at 15,000 × g for 1 minute. Synaptosomal viability was assessed using the MTT assay (Mungarro-Menchaca et al. 2002).

Determination of Reduced Glutathione (GSH)

Following incubation with the test compounds, synaptosomes were centrifuged at 400 × g for 3 minutes. The resulting pellet was treated with 5% trichloroacetic acid, thoroughly vortexed, and left to sit on ice for 10 minutes. This was followed by centrifugation at 8,000 × g for 10 minutes to separate the supernatant. The obtained supernatant was stored at −20 °C until analysis.

Prior to analysis, the samples were neutralized by adding 5 M NaOH. GSH levels were then quantified spectrophotometrically at 412 nm using Ellman’s reagent (DTNB) (Robyt et al. 1971). This approach allowed for precise determination of reduced glutathione levels in the synaptosomal preparations.

Tert-Butyl Hydroperoxide (t-BuOOH)-Induced Oxidative Stress in rat brain mitochondria

For experimental purposes, the mitochondria were incubated with 75 μM t-BuOOH for 1 h, following the methodology described by Karlsson et al. (2000), to evaluate the effects of induced oxidative stress.

When administered alone, the substances were incubated with the brain sub-cellular fractions (synaptosomes, brain mitochondria and brain microsomes) for 1 h. In combination with the neurotoxic agents, initially, we made a 30-minute pre-incubation with the substances alone, and after that, we administered the neurotoxic agent and incubated the combination for 1 h.

Measurement of GSH content in brain mitochondria

Following the incubation of mitochondria, 0.04% 5,5’-dithiobis (2-nitrobenzoic acid) (DTNB) was added to the mitochondrial suspensions in 0.1 M phosphate buffer (pH 7.4). The reaction resulted in the formation of a yellow product, and the absorbance of this product was measured at 412 nm to quantify the glutathione (GSH) content, as outlined in the method by Shirani et al. (2019).

Determination of malondialdehyde (MDA) production in brain mitochondria (Shirani et al. 2019)

To the mitochondria was added 0.3 ml of 0.2% thiobarbituric acid and 0.25 ml of sulfuric acid (0.05 M), and the mixture was boiled for 30 minutes. After boiling, the tubes were placed on ice, and 0.4 ml of n-butanol was added to each, then centrifuged at 3,500 × g for 10 minutes. The amount of MDA was determined spectrophotometrically at 532 nm.

Iron/ascorbate-induced lipid peroxidation (LPO) in brain microsomes

Non-enzyme-induced lipid peroxidation was induced with 20 μM ferrous sulfate solution and 0.5 mM ascorbic acid solution for 1 hour (Mansuy et al., 1986).

Determination of MDA in brain microsomes (Mansuy et al. 1986)

After the completion of incubation of the microsomes with the substances and toxic agent, the reaction was stopped by the addition of 0.5 ml of 20% trichloroacetic acid followed by 0.5 ml of 0.67% thiobarbituric acid. The ongoing reactions are associated with the formation of a coloured complex between the malondialdehyde formed and thiobarbituric acid. The determination of MDA was measured spectrophotometrically at 535 nm. A molar extinction coefficient of 1.56 × 105 M-1 cm-1 was used for the calculation.

FeSO4/Ascorbic Acid-Induced Lipid Peroxidation (LPO) in Isolated Liver Microsomes

Isolated microsomes were pre-incubated with NLC, BRB, and NLC-B for 30 min, and after that, lipid peroxidation (LPO) was initiated by adding 20 μM iron sulfate and 0.5 mM ascorbic acid for 60 minutes at 37 °C (Mansuy et al. 1986).

Malondialdehyde (MDA) Assay in liver microsomes

Lipid peroxidation is quantified by assessing the in vitro formation of thiobarbituric acid-reactive substances, specifically malondialdehyde (MDA), a by-product of this process. When MDA interacts with 2-thiobarbituric acid (TBA), a colored complex is formed, consisting of one molecule of MDA and two molecules of TBA. The intensity of this color complex correlates directly with the concentration of MDA present. After incubation, 0.5 ml of the liver microsomes were mixed with 0.5 ml 25% trichloroacetic acid (TCA) and 0.5 ml 0.67% thiobarbituric acid (TBA). After boiling the mixture for 30 min and then cooling, the samples were centrifuged for 5 minutes at 2,000 × g. The malondialdehyde (MDA) levels were measured to assess the extent of lipid peroxidation spectrophotometrically at a wavelength of 532 nm (Kim et al. 1997).

Statistical analysis

Statistical analyses were conducted using GraphPad Prism 8 Software (version 8, GraphPad Software, La Jolla, California, USA). The data were first analysed using a one-way ANOVA, followed by Dunnett’s multiple comparisons post-test to compare control and treatment groups. Statistical significance was determined with p-values of *p ≤ 0.05, **p ≤ 0.01, and ***p ≤ 0.001.

For the analysis of data obtained from brain microsomes, synaptosomes, and mitochondria, the statistical program “MEDCALC” was used. Results were presented as mean ± SEM from three independent experiments. The significance of differences was evaluated using the non-parametric Mann-Whitney test. p-values of p < 0.05, p < 0.01, and p < 0.001 were considered statistically significant. A two-way ANOVA was performed to compare the matched berberine and NLC-B treatment groups at each concentration. A Tukey post hoc test was applied to compare each pair of groups. Additional statistical analysis was performed to compare the different groups, NLC-B and BRB in different models of oxidative stress and lipid peroxidation. Statistical significance was determined using the Holm-Sidak method, with alpha = 0.05. Each row was analysed individually, without assuming a consistent standard deviation.

Results

Nanoparticles preparation and characterization

Nanostructured lipid carriers (NLCs) were successfully prepared and loaded with berberine. Their physicochemical attributes are presented in Table 1.

Table 1.
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Size, polydispersity index (PDI), zeta potential, encapsulation efficiency (EE, %) and loading capacity (LC, %) of the drug delivery systems immediately upon preparation and after 6 months of storage at room temperature under dark (mean ± SD, n = 3); *statistically significant difference (p < 0.05) with 0 months.

Sample Time point Size, nm PDI Zeta potential, mV EE, % LC, %
NLC 0 months 168.1 ± 2.3 0.184 ± 0.02 -32.8 ± 1.1 – –
6 months 176.5 ± 1.8* 0.246 ± 0.00* -26.8 ± 1.3* – –
NLC-B 0 months 158.2 ± 1.8 0.305 ± 0.01 -30.7 ± 0.8 97.7 ± 2.3 3.46 ± 1.7
6 months 168.9 ± 1.4* 0.221 ± 0.00* -31.4 ± 0.4 90.8 ± 1.5* 3.22 ± 0.9*

There was a significant change in the hydrodynamic diameter of the nanoparticles upon mixing with SNF with pH 6.4 (Fig. 1). The water-diluted samples had an average size of 176.5 ± 2.3 nm and 168.9 ± 1.7 nm for the NLC and NLC-B, respectively. The dilution with SNF correspondingly led to 124.3 ± 3.0 nm and 140.65 ± 3.3 nm. The zeta potential also increased significantly from -32.8 ± 1.1 to -2.32 ± 0.38 for the NLC and from -30.7 ± 0.8 to -5.10 ± 2.6 for the NLC-B.

Figure 1. 

Hydrodynamic diameter by intensity for the empty (A, C) and NLC-B (B, D) in water (A, B) and in SNF (C, D).

The in vitro release study conducted in SNF by the dialysis-bag method revealed improved solubility with prolonged release over time, as shown in Fig. 2.

Figure 2. 

In vitro release profiles of free berberine (BRB) and NLC-B in simulated nasal fluid (SNF) at 37 ± 0.5 °C; (mean ± SD; n = 3).

In vitro study on rat brain synaptosomes

An in vitro evaluation was conducted to assess the potential toxicity, antioxidant, and neuroprotective effects of NLC, BRB and NLC-B in a 6-OHDA-induced toxicity model using rat brain synaptosomes.

Effects of NLC, free BRB and NLC-B on synaptosomal viability and GSH levels in isolated rat brain synaptosomes

The primary parameters used to characterize the functional-metabolic status of brain synaptosomes were synaptosome viability and reduced glutathione (GSH) levels (Fig. 3A, B). The results demonstrated a statistically significant, concentration-dependent neurotoxic effect of empty nanoparticles, free BRB and NLC-B when applied alone, compared to the control untreated brain synaptosomes. Notably, NLC-B nanoparticles exhibited lower toxicity than free BRB. Empty nanoparticles decreased the synaptosome viability in a concentration-dependent manner. Thus, at an NLC concentration of 10 μg/mL, the synaptosome viability was reduced by 10 %, while NLC 50 µg/mL caused 20% reduction. Increasing the concentrations up to 500 µg/mL resulted in a 50% reduction of synaptosome viability (Fig. 3A). Similarly, to the empty NLC, free BRB significantly reduced synaptosome viability in a dose-dependent manner. For example, at concentrations of 1 µM and 5 µM free BRB, the viability decreased by 10%, and 15%, respectively, while at 50 µM, synaptosomal viability was reduced by 50% compared to the untreated control. In contrast, NLC-B exhibited lower toxicity. At 1 µM, no toxicity was observed; at 5 µM synaptosome viability was reduced only by 10%, while 50 µM caused a 40% reduction, compared to the untreated control synaptosomes. These findings highlight that while free BRB exhibited some toxicity on synaptosomes, the encapsulating berberine in nanostructured lipid carriers (NLC-B) reduced its toxic effects.

Figure 3. 

Effects of NLC, BRB and NLC-B on synaptosomal viability (A) and reduced glutathione levels (GSH) (B) in isolated rat brain synaptosomes. The concentrations of NLC are 10, 50, 100, 250 and 500 µg/ml and the concentrations of berberine (free and loaded in NLC) are 1, 5, 10, 25 and 50 µM *p < 0.05, **p < 0.01, ***p < 0.001 vs. control (non-treated synaptosomes) # p < 0.05, ## p < 0.01 comparison between different treated groups. Data are presented as mean ± SD from three independent experiments (n = 3).

A similar trend was observed for the reduced glutathione (GSH) levels, where treatment with the NLC, BRB, and NLC-B resulted in a concentration-dependent and statistically significant decrease. Notably, the reduction in GSH levels was least pronounced in loaded NLC-B, followed by free BRB, while the highest toxicity was observed with empty NLC (Fig. 3B).

Neuroprotective effects in a model of 6-OHDA-induced toxicity

In the next stage, the potential neuroprotective and antioxidant effects of the studied samples were evaluated using an in vitro model of 6-OHDA-induced damage. This in vitro model effectively mimics the neurodegenerative processes associated with Parkinson’s disease (PD). The metabolism and oxidation of dopamine generate reactive oxygen species (ROS) and reactive quinones, processes catalysed by the monoamine oxidase B (MAO-B) enzyme. These reactions contribute to dopamine-induced neurotoxicity and the progressive neurodegeneration observed in PD (Stokes et al. 2002).

The administration of 6-hydroxydopamine (6-OHDA) caused a statistically significant reduction by 50% in both synaptosomal viability and GSH levels, compared to the control – untreated brain synaptosomes (Fig. 4A, B). Both free BRB and loaded NLC-B exhibited a statistically significant, concentration-dependent neuroprotective effect compared to the 6-OHDA-treated group. Notably, NLC-B demonstrated a superior neuroprotective effect compared to free BRB. At the highest tested concentrations of 25 and 50 µM, BRB preserved synaptosomal viability by 40%, while GSH levels – by 30% and 40%, respectively. In comparison, NLC-B at the same concentrations preserved synaptosomal viability by 40% and 60%, and GSH levels – by 30% and 60%, respectively.

Figure 4. 

Neuroprotective effects of NLC, BRB, and NLC-B on synaptosomal viability (A) and reduced glutathione (GSH) levels (B) in a 6-OHDA-induced toxicity model in isolated rat brain synaptosomes. The concentrations of berberine (free and loaded in NLC) are 1, 5, 10, 25 and 50 µM ***p < 0.001 vs. control (non-treated synaptosomes); +p < 0.05, ++p < 0.01, +++p < 0.01 vs. 6-OHDA, # p < 0.05 comparison between different treated groups. Data are presented as mean ± SD from three independent experiments (n = 3).

In vitro study in isolated rat brain mitochondria

Effects of NLC, BRB and NLC-B on MDA and GSH levels

Mitochondrial dysfunction is a key contributor to neuronal damage and disease progression. Evaluation of the impact of the tested compounds on mitochondrial health provides valuable insights into their potential neuroprotective or neurotoxic properties. Thus, we investigated the effects of NLC, BRB and NLC-B on isolated rat brain mitochondria. Malondialdehyde (MDA) production and reduced glutathione (GSH) levels serve as key indicators of the functional-metabolic status of brain mitochondria. By assessing both MDA and GSH levels, we found that the treatment with empty NLC and BRB induced a statistically significant, concentration-dependent neurotoxic effects, compared to the control untreated brain mitochondria. Interestingly, NLC-B demonstrated lower toxicity than free BRB further highlighting the potential mitigating effects of nanoparticle encapsulation (Fig. 5). Evaluation of the effects on MDA production, we noticed that the empty NLC induced the highest levels of MDA production. For instance, NLC treatment gradually increased MDA by 20% (10 µg/mL) and by 130 % at 500 µg/mL (Fig. 5A). NLC-B and free BRB showed a gradual increase in MDA content by 10 % and 20 %, respectively (at 1 µM) and by 20 % and 30 %, respectively (at 5 µM). At the highest tested concentration 50 µM, NLC-B and free BRB caused significant increase in MDA, by 70% and 80%, respectively.

Figure 5. 

Effects of NLC, BRB and NLC-B on MDA levels (A) and level of GSH (B) in isolated rat brain mitochondria. The concentrations of NLC are 10, 50, 100, 250 and 500 µg/ml and the concentrations of berberine (free and loaded in NLC) are 1, 5, 10, 25 and 50 µM *p < 0.05, **p < 0.01, ***p < 0.001 vs. control (non-treated synaptosomes) # p < 0.05, ## p < 0.01, ### p < 0.001 comparison between different treated groups. Data are presented as mean ± SD from three independent experiments (n = 3).

After evaluation of the effects on mitochondrial GSH levels, we found that loaded NLC-B exhibited lower toxicity compared to free unloaded BRB. No significant reduction in GSH levels was observed at 1 µM NLC-B, while free BRB caused a 10% decrease. At 5 µM, GSH levels declined by 10% by NLC-B and by 15% with free BRB. At 50 µM, the GSH reductions were 35% and 40%, respectively. The empty NLC showed the most pronounced GSH depletion, with reductions of 15% at concentration 10 µg/mL to 40% at the highest tested concentration 500 µg/mL, compared to the untreated control (Fig. 5B). The analysis of the results has shown that all tested samples induced oxidative stress to some degree. Nevertheless, NLC-B exhibited reduced toxicity compared to free BRB. This highlights the potential benefits of encapsulating BRB in lipid nanoparticles to mitigate its harmful effects, especially at lower concentrations.

Protective effects in a model of tert-butyl hydroperoxide (t-BuOOH) induced oxidative stress

A well-established model for studying oxidative stress involves the use of tert-butyl hydroperoxide (t-BuOOH), a compound that undergoes both mitochondrial and microsomal metabolism. The breakdown of t-BuOOH leads to the generation of reactive oxygen species (ROS) through distinct metabolic pathways, depending on the cellular environment. In isolated mitochondria and intact cells, t-BuOOH undergoes β-cleavage, leading to the formation of a highly reactive methyl radical. These radicals play a crucial role in initiating lipid peroxidation, a process that disrupts cellular membrane integrity and contributes to oxidative damage. Additionally, the presence of these free radicals leads to a significant reduction in intracellular levels of reduced glutathione (GSH), a key antioxidant responsible for neutralizing oxidative stress. When administered alone, tert-butyl hydroperoxide (t-BuOOH) led to a statistically significant increase in MDA production by 250% and a reduction in GSH levels by 50%, compared to the untreated control brain mitochondria (Fig. 6). In a model of t-BuOOH-induced oxidative stress, both free BRB and NLC-B demonstrated statistically significant, concentration-dependent antioxidant effects compared to the positive controls (mitochondria treated with t-BuOOH). Notably, NLC-B exhibited superior neuroprotective and antioxidant properties compared to free BRB (Fig. 6). At the lowest concentration of 1 µM, NLC-B reduced MDA production by 26%, while free BRB achieved a 14% reduction, compared to the toxic agent t-BuOOH. At 5 µM, NLC-B decreased MDA production by 34%, whereas free BRB reduced it by 20%. At 10 µM, the reduction was 43% for NLC-B and 29% for free BRB. At 25 µM, NLC-B led to a 49% decrease, while free BRB reduced MDA levels by 43%. The highest tested concentration, 50 µM, resulted in a 54% reduction for NLC-B and a 46% reduction for free BRB (Fig. 6A). While evaluating the effects of the tested samples on GSH levels, we found that at 1–5 µM, NLC-B preserved GSH levels by approximately 20%, and the free BRB showed similar results. At 10 µM and 25 µM, NLC-B preserved GSH by 30%, whereas free BRB maintained a 20% preservation at both concentrations. At 50 µM, NLC-B preserved GSH levels by 50%, while free BRB preserved them by 40% (Fig. 6B).

Figure 6. 

Neuroprotective effects of NLC, BRB and NLC-B on MDA levels (A) and level of GSH (B) in a t-BuOOH-induced toxicity model in isolated rat brain mitochondria. The concentrations of berberine (free and loaded in NLC) are 1, 5, 10, 25 and 50 µM *p < 0.05, **p < 0.01, ***p < 0.001 vs. control (non-treated synaptosomes); +p < 0.05, ++p < 0.01 vs. 6-OHDA) # p < 0.05, ## p < 0.01, comparison between different treated groups. Data are presented as mean ± SD from three independent experiments (n = 3).

These findings indicate that both free BRB and NLC-B exhibited antioxidant and protective effects. Nevertheless, the nanoparticle formulation NLC-B consistently demonstrated greater efficacy in reducing oxidative damage and preserving cellular defence mechanisms.

Effects of non-loaded BRB and loaded NLC-B in a model of iron/ascorbate-induced peroxidation on isolated rat microsomes

Protective effects on isolated rat brain microsomes

The isolated rat brain microsomes represent a well-established in vitro model for assessing the pharmacological properties and toxicity of various substances. This neuronal model provides valuable insight into the direct effects of compounds on neuronal function and cellular metabolism. (Ahmad and Liu 2020). Iron/ascorbate-induced lipid peroxidation is often used in experimental setups to evaluate the antioxidant properties of different compounds and their potential to prevent or mitigate oxidative damage. By measuring the extent of lipid peroxidation and monitoring the levels of key biomarkers, such as malondialdehyde (MDA), it is possible to assess the effectiveness of the tested substances in conditions of oxidative stress.

The iron/ascorbate treatment caused a significant increase in MDA production by 250%, compared to the control untreated brain microsomes (Fig. 7A). Both NLC-B and free BRB demonstrated a concentration-dependent, statistically significant antioxidant effect. Interestingly, NLC-B exhibited a stronger and statistically significant antioxidant effect than free BRB. For example, NLC-B (50 µM) reduced MDA production by 61%, while free berberine, at the same concentration, reduced MDA production by 49%, compared to the positive control (iron/ascorbate treated microsomes). These results suggest that while both loaded NLC-B and non-loaded BRB exhibited antioxidant effects, the NLC-B nanoparticles were more effective in reducing lipid peroxidation, especially at higher concentrations (Fig. 7A).

Figure 7. 

Protective effects of BRB and NLC-B on MDA levels in a Fe2+/AA-induced toxicity model in isolated rat brain microsomes (A) and rat liver microsomes (B). The concentrations of berberin (free and loaded in NLC) are 1, 5, 10, 25 and 50 µM. ***p < 0.001 vs. control non-treated microsomes; +p < 0.05, ++p < 0.01, +++p < 0.001 vs. 6-OHDA) # p < 0.05, ### p < 0.001 comparison between different treated groups. Data are presented as mean ± SD from three independent experiments (n = 3).

Protective effects on isolated rat liver microsomes

In this study, liver microsomes were employed as an in vitro model to simulate cell membranes, as they closely mimic the phospholipid-rich environment of biological membranes (Agarwal et al. 1992). This approach allows controlled assessment of the oxidative damage and evaluation of antioxidant effects of different substances under experimental conditions. Iron/ascorbate treatment resulted in statistically significant increase in MDA production by 250 % compared to the control untreated liver microsomes (Fig. 7B). In this model of non-enzymatic lipid peroxidation, NLC-B and free BRB demonstrate a statistically significant and concentration-dependent antioxidant effects (Fig. 7B). The most significant antioxidant effects of free BRB were demonstrated at concentrations from 5 µM to 50 µM, where MDA production was reduced by 25% and by 46%, respectively. Notably, NLC-B exhibits a superior antioxidant activity compared to free BRB. For example, 5 µM of NLC-B reduced MDA production by 43% in a statistically significant manner, whereas 50 µM by 59% compared to the untreated control (Fig. 7B).

Discussion

Nanostructured lipid nanocarriers (empty and berberine loaded) were prepared by the melt-emulsification method. The method for their preparation was robust and reproducible (Stefanova et al. 2025). The obtained nanocarriers fall in the suitable size range (Table 1) for potential intranasal delivery (Costa et al. 2019). According to available data, an NLC with sizes smaller than 200 nm and with PDI less than 0.3 are most appropriate for the nasal administration route (Costa et al. 2021a)Their negative zeta potential renders them with good physicochemical stability (Onugwu et al. 2023). In addition, there are evidence supporting the suitability of negatively charged nanocarriers for better overcoming the mucus layer. This layer contains sialic and sulfonic acids which determine its negative zeta potential. Hence, positively charged particles tend to be trapped in the mucus layer and cannot reach the epithelium (Dünnhaupt et al. 2015).

The size and zeta potential remain relatively stable over time due to the good miscibility between the lipids as previously reported (Jeitler et al. 2024). Even though a statistically significant difference in the size and PDI is observed (Table 1), the size and PDI still fall in the appropriate range. It can be considered that these change in parameters possibly would not affect significantly the behaviour of the nanocarrier upon instillation. In the case of zeta potential siginificant difference is observed only in the case of the empty NLC. It is also evident that the drug is retained in high amount within the delivery system over 6 months of storage at room temperature. These data confirm the inherent high drug loading and limited expulsion during storage of the NLCs (Elmowafy and Al-Sanea 2021). The storage at room temperature, however results in significant decrease in the encapsulation efficiency (Table 1).

The evaluation of the nanoparticles’ physicochemical properties after dilution with simulated nasal fluid (SNF) showed consistent features. The observed hydrodynamic size is decreased which could be expected due to the presence of salts in the SNF which is associated with the so-called hydrophobic effect and a thinner hydration shell is observed (Gordillo-Galeano and Mora-Huertas 2021). This suggests that the proposed nanosized drug delivery system has potential for staying stable when mixed with nasal secretion. Furthermore, the mixing with SNF resulted in a significant decrease in the absolute value of the zeta potential. This could be attributed to the neutralizing effect of the present ions. It can be hypothesized that the overcoming of the mucus layer and the intracellular transport could additionally benefit (Dünnhaupt et al. 2015). Nevertheless, there are still additional optimizations needed in order to achieve optimal residence time and appropriate permeability characteristics which were not the aim of the current work.

The in vitro release study was conducted over 24 h (Fig. 2) as a standard dissolution duration for nasal delivery (Costa et al. 2021b; Yasir et al. 2022) that could be used in the future for comparison with other results. The investigation of the release behaviour suggested improved solubility of the drug when loaded in the NLC-B. The burst initial release suggests possibility for fast onset of absorption prior to elimination by the nasal mucocilliary clearance (Sallam et al. 2016). In addition, prolonged release over 24 h was evident which could achieve more persistent concentration without significant alterations. However, this concept could be proven only by in vivo experiments, which could be a subject of investigation in future once the formulation deems suitable.

In order to evaluate the toxicity, neuroprotective, and antioxidant potential of berberine alone or encapsulated within NLCs a battery of isolated subcellular brain fractions, such as synaptosomes, microsomes and mitochondria were used. The findings highlight the neuroprotective properties of BRB, emphasizing the beneficial impact of its encapsulation in NLC. Both synaptosomal viability and GSH levels are crucial indicators of synaptosomal function with GSH playing a central role in synaptosomal protection against oxidative damage. The in vitro assessment of the effects of NLC, BRB and NLC-B revealed that they exhibited a concentration-dependent reduction in synaptosomal viability. The empty NLC nanoparticles demonstrated the highest toxicity, followed by free BRB, while the loaded NLC-B showed the lowest toxicity. These findings suggest that while BRB alone possesses inherent toxicity at higher concentrations, its encapsulation in NLCs effectively mitigates this effect. Similarly, NLC-B displayed the least pronounced reduction in GSH levels (measured as a toxicity parameter for oxidative stress) in comparison to the empty NLC and BRB. This suggests that the prepared NLC-B might provide a better protection of potentially mitigating berberine’s prooxidative effects in brain synaptosomes.

6-OHDA model, used to simulate in vitro the pathology mechanisms associated with Parkinson’s disease (PD) (Negahdar et al. 2015) further emphasized the neuroprotective potential of NLC-B. In this model, 6-OHDA treatment alone caused a significant reduction in synaptosomal viability and GSH levels, indicating neurotoxicity impairment. Both free BRB and NLC-B exhibited concentration-dependent neuroprotective effects, but it should be noted that NLC-B demonstrated a superior protection, compared to free BRB. These results align with previous studies highlighting the neuroprotective effects of BRB in vitro and in vivo. Negahdar et al. demonstrated that BRB in dose of 50 mg/kg can alleviate behavioural and neuronal damage in 6-OHDA-induced rat model of Parkinson’s disease, showing significant reduction of motor deficits and neuronal loss (Negahdar et al. 2015). Jinbum Bae et al. found that BRB protects neuronal cells from 6-OHDA-induced cell death by reducing ROS, caspase-3 activation, and upregulating HO-1 via Nrf2. BRB also activated PI3K/Akt and p38, contributing to neuroprotection, suggesting its potential as a therapy for dopaminergic neuronal diseases (Bae et al. 2013; Zhang et al. 2017). Importantly, the enhanced protective effects of NLC-B can be attributed to the improved stability, solubility, and bioavailability of BRB after encapsulation in nanostructured lipid carriers NLC, which may allow more effective targeting and greater efficacy. Overall, while free BRB exhibited significant neuroprotective effects, the loaded NLC-B provided superior protection, as shown by higher synaptosomal viability and better preservation of GSH. These findings suggest that encapsulation of berberine in NLC enhances its pharmacological efficacy. The prepared NLC-B offer a promising approach for minimizing BRB toxicity, while maximizing its neuroprotective effects in the context of neurodegenerative diseases, such as Parkinson’s disease.

Considering the essential role of mitochondrial function in cellular homeostasis, especially in dopaminergic neurons, and its involvement in the neuropathogenic mechanisms of neurodegenerative diseases (Maiti et al. 2017; Mani et al. 2023), our study also assessed the impact of the NLC-B on mitochondrial function. This was done by measuring malondialdehyde (MDA) production and glutathione (GSH) levels in isolated rat brain mitochondria. MDA, a by-product of lipid peroxidation, serves as a key marker of oxidative stress, while GSH levels reflect the antioxidant capacity of the cells. Consistent with previous findings, empty nanoparticles induced the highest pro-oxidant effects, followed by free BRB. In contrast, NLC-B exhibited significantly lower toxicity, particularly at higher concentrations, suggesting that encapsulating berberine in NLCs reduces its harmful effects on cellular function. Since tert-butyl hydroperoxide (t-BuOOH) is widely recognized as a model for inducing oxidative stress due to its metabolism within mitochondrial and microsomal systems (Marí et al. 2009), we further investigated the formulations under t-BuOOH-induced oxidative stress conditions. Both free BRB and NLC-B exhibited significant neuroprotective and antioxidant effects in this experimental model. However, NLC-B outperformed free BRB in reducing MDA production and preserving GSH levels, further highlighting the enhanced antioxidant potential of encapsulated NLC-B. A possible explanation could be that nanoparticle encapsulation might enhance the bioavailability and cell penetration of BRB, leading to more effective mitigation of mitochondrial dysfunction and oxidative damage. In summary, encapsulating BRB in NLCs improves its antioxidant properties, making it more effective in combating oxidative damage compared to free berberine alone.

The assessment of the effects of NLC, BRB, and NLC-B on isolated rat brain microsomes provides valuable insights into their antioxidant properties. Brain microsomes serve as an excellent model for evaluating the toxicity of substances, as they closely represent the functional properties of the brain’s cellular membranes (Harry et al. 1998). In our study, we observed that both NLC and BRB, administered alone, induced an increase in oxidative stress, which is evidenced by the elevated production of malondialdehyde (MDA), a biomarker of lipid peroxidation. In contrast, the encapsulated NLC-B demonstrated a significantly reduced pro-oxidant effect, particularly at higher concentrations, suggesting a protective role in mitigating oxidative damage.

A widely recognized model for studying lipid peroxidation is the iron/ascorbate-induced peroxidation system (Hassel et al. 2022). This model mimics oxidative stress by utilizing a combination of iron (Fe2+) and ascorbate (vitamin C), which together catalyse the generation of reactive oxygen species (ROS), particularly hydroxyl radicals. These free radicals initiate the oxidation of lipids, leading to the formation of lipid peroxides, which serve as biomarkers of oxidative damage. This system is especially relevant in studying the mechanisms underlying various neurodegenerative diseases, as the oxidative damage to cellular membranes and organelles is a key feature of the pathophysiology. Furthermore, the antioxidant potential of NLC-B was confirmed in an iron/ascorbate-induced lipid peroxidation model in brain and liver microsomes (Iqbal et al. 1998). Both free BRB and NLC-B demonstrated significant antioxidant effects. However, NLC-B exhibited superior efficacy in reducing MDA production compared to free BRB at the same concentrations. At 50 µM, NLC-B reduced MDA production by 61%, while free BRB only reduced it by 49%, indicating that nanoparticle encapsulation not only enhanced the bioavailability of BRB, but also improved its ability to scavenge free radicals. This improvement can be attributed to the controlled release mechanism of NLC-B, which facilitates a more sustained antioxidant effect. The encapsulation likely prevents rapid degradation or potential toxicity of BRB, resulting in a more consistent protective action. These findings support the hypothesis that the encapsulation of BRB in nanoparticles not only enhances its antioxidant properties but also reduces its potential for inducing oxidative stress, as seen in the brain microsome and lipid peroxidation models.

Similar results were observed in liver microsomes, where the combination of iron and ascorbate generated significant oxidative stress, leading to increased MDA production. NLC-B exhibited superior antioxidant activity compared to free berberine, further reinforcing the benefits of nanoparticle encapsulation. At 50 µM, NLC-B reduced MDA production by 59%, while free berberine achieved only a 46% reduction. This is consistent with the notion that NLC-B provides better protection against oxidative damage, likely due to more efficient cellular uptake and enhanced distribution in target tissues. In liver microsomes, both NLC-B and free BRB showed a concentration-dependent reduction in MDA production. However, the NLC-B formulation consistently outperformed free berberine, confirming that encapsulation improves the bioavailability and antioxidant efficacy of BRB. This result is in line with previous studies highlighting the role of nanoparticles in enhancing the pharmacological properties of bioactive compounds by improving their solubility, stability, and controlled release (Agarwal et al. 1992). The antioxidant mechanism of berberine-loaded nanoparticles and free berberine is most likely linked to their ability to scavenge free radicals and to stabilize cellular membranes. Free radicals, such as reactive oxygen species (ROS), are known to induce oxidative damage, which can compromise the structural integrity of membranes. By neutralizing these reactive species, both forms of berberine can protect against lipid peroxidation and maintain membrane stability.

Neurotrophins such as nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), and neurotrophins 3, 4, and 5 promote neuronal survival by binding to Trk receptors and activating signalling pathways that prevent cell death (Yamamoto et al. 1993). Research on neurodegenerative diseases indicates that BRB supports neuroprotection by regulating neurotrophins levels (Uddin et al. 2020). At nanomolar concentrations, it enhances cell survival by activating PI3K/Akt/Nrf2 signalling and suppressing p53 and cyclin D1 (Lee et al. 2010). Additionally, BRB stimulates the production of neurotrophic mediators, including tumour necrosis factor-alpha (TNF-α), nitric oxide (NO), prostaglandin E2 (PGE2), and interleukin-1 (IL-1) (Lee et al. 2010).

Conclusion

This study demonstrates that both free berberine and NLC-B exhibit neuroprotective effects in different in vitro models of neurotoxicity, with NLC-B showing superior efficacy. While all tested formulations exhibited some level of toxicity, the encapsulation of berberine in nanostructured lipid carriers significantly reduced its harmful effects while enhancing its neuroprotective and antioxidant properties. The improved safety profile of NLC-B might be explained by its enhanced solubility, bioavailability, and controlled release after encapsulation. These findings suggest that NLC-B has strong potential as a pharmacological strategy for neurodegenerative diseases by mitigating oxidative stress, preserving mitochondrial function, and improving synaptosomal viability. Future studies should be focused on in vivo evaluations to further confirm these findings and explore the potential of NLC-B as a potential treatment strategy for neurodegenerative impairments.

Additional information

Conflict of interest

The authors have declared that no competing interests exist.

Ethical statements

The authors declared that no clinical trials were used in the present study.

The authors declared that no experiments on humans or human tissues were performed for the present study.

The authors declared that no informed consent was obtained from the humans, donors or donors’ representatives participating in the study.

The authors declared that no experiments on animals were performed for the present study.

The authors declared that no commercially available immortalised human and animal cell lines were used in the present study.

Funding

The authors would like to acknowledge the financial support by the European Union- Next GenerationEU, through the National Recovery and Resilience Plan of the Republic of Bulgaria, project № BG-RRP-2.004-0004-C01.

Radostina Bogdanova is gratefully acknowledged for funding the in vitro cytotoxicity tests, sponsored by the Medical Science Council of the Medical University of Sofia under the project “Young Researcher 2024,” Д-107/29.05.2024.

Author contributions

Conceptualization, D.S. and M.S.; methodology, M.S. and D.S.; investigation, R.B., M.K-B, V.Tz., T.P.; Y.Y. data curation, B.Tz.; writing – D.S., M.S. original draft preparation, D.S.; writing – review and editing, D.S., Ch.V, T.P, V.Tz.; visualization, B.Tz; supervision, M.S.; All authors have read and agreed to the published version of the manuscript.

Author ORCIDs

Teodora Popova https://orcid.org/0000-0002-6690-6740

Christina Voycheva https://orcid.org/0000-0002-6536-4898

Marta Slavkova https://orcid.org/0000-0001-5335-461X

Data availability

All of the data that support the findings of this study are available in the main text.

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