Solid lipid nanoparticles loaded with a novel indole-based multitarget ligand: preparation, characterization, and neuroprotective evaluation in an H₂O₂-induced SH-SY5Y cell model

Solid lipid nanoparticles loaded with a novel indole-based multitarget ligand: preparation, characterization, and neuroprotective evaluation in an H₂O₂induced SH-SY5Y cell model Christina Voycheva1, Teodora Popova1, Marta Slavkova1, Borislav Tzankov1, Boris Stoyanov1, Denitsa Stafanova1, Violina Angelovа1 1 Faculty of Pharmacy, Medical University of Sofia, Sofia, Bulgaria Corresponding author: Christina Voycheva (hvo[email protected]u-sofia.bg) Received 29 September 2025♦ Accepted 23 October 2025♦ Published 18 November 2025 Citation: Voycheva C, Popova T, Slavkova M, Tzankov B, Stoyanov B, Stafanova D, Angelovа V (2025) Solid lipid nanoparticles loaded with a novel indole-based multitarget ligand: preparation, characterization, and neuroprotective evaluation in an H₂O₂-induced SH-SY5Y cell model. Pharmacia 72: 1–13. https://doi.org/10.3897/pharmacia.72.e173489 Abstract A newly synthesized indole-based N-benzylpiperidine derivative (3a), acting as a multitarget-directed ligand with acetylcholinesterase inhibitory activity, was encapsulated into solid lipid nanoparticles (SLNs) to improve neuroprotective potential and enable intranasal brain delivery in future in vivo studies. Lipid prescreening was performed, and a spectrophotometric method for the quantification of 3a was developed and validated. SLNs were prepared using the nano-template engineering technique and evaluated for particle size, zeta potential, drug entrapment efficiency, in vitro release, and stability. The optimized formulation (3a-SLNs) was further characterized by transmission electron microscopy (TEM), X-ray powder diffraction (XRD), and differential scanning calorimetry (DSC). The nanoparticles exhibited favorable physicochemical properties, releasing 52% of the drug over 24 h. Permeability studies indicated improved cellular uptake of 3a upon SLN encapsulation. The formulation remained stable for 6 months under refrigerated (4 ± 2 °C) and room temperature (25 ± 2 °C; 60 ± 5% RH) conditions. The neuroprotective potential of 3a-SLNs was evaluated in an H₂O₂-induced oxidative stress model in SH-SY5Y neuronal cells using the MTT assay. Cells were pretreated with free 3a (0.1–50 µM) for 90 min before H₂O₂ exposure (1 mM). Hydrogen peroxide induces oxidative damage via reactive hydroxyl radicals, affecting cellular lipids, proteins, and DNA. 3a-SLNs exhibited superior protective effects compared with free 3a, indicating that nanoparticle encapsulation enhances intracellular availability and antioxidant defense. These findings highlight SLN-encapsulated 3a as a promising intranasal nanocarrier system for Alzheimer’s disease therapy, which will be further investigated in in vivo models. By improving brain delivery potential and reducing oxidative stress–induced neuronal injury, 3a-SLNs offer a strategy to overcome pharmacokinetic limitations of conventional compounds and enhance neuroprotective efficacy. Keywords Alzheimer’s disease, multitarget ligand 3a, neuroprotection, nose-to-brain delivery, oxidative stress, SH-SY5Y cells, solid lipid nanoparticles Copyright Voycheva C et al. 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. Pharmacia 72: 1–13 DOI 10.3897/pharmacia.72.e173489 Research Article Voycheva C et al.: SLNs loaded with a novel indole-based multitarget ligand2 Introduction Alzheimer’s disease (AD) is a progressive neurodegenerative disorder characterized by cognitive decline, memory loss, and neuronal death (Cardinali et al. 2010). Among the major pathological mechanisms underlying AD are cholinergic dysfunction, oxidative stress, and accumulation of amyloid-beta (Aβ) peptides. Cholinergic deficits are associated with decreased acetylcholine (ACh) signaling, primarily due to altered activity of acetylcholinesterase (AChE) and butyrylcholinesterase (BChE), which hydrolyze ACh. In AD, AChE activity is often unchanged or decreased, while BChE activity may increase, contributing to impaired cholinergic transmission (Fernández-Bolaños and López 2022). Concurrently, Aβ peptides accumulate due to imbalances in production and clearance, forming neurotoxic oligomers that trigger oxidative stress, lipid peroxidation, and neuronal apoptosis (Li et al. 2020). Donepezil (DNPZ) is a selective, noncompetitive, and rapidly reversible acetylcholinesterase inhibitor. Melatonin (MEL) modulates the Aβ production/ clearance balance and reduces Aβ neurotoxicity (Ramos et al. 2017; Li et al. 2020; Verma et al. 2023). Molecules containing a melatonin frame and a donepezil remnant show prominent inhibitory activity against acetylcholinesterase (Luo et al. 2013; Gulcan and Kosar 2022; Pravin and Jozwiak 2022; Eissa et al. 2023). A recently developed indole-based N-benzylpiperidine derivative, 3a (N’-[(E)- (1-benzylpiperidin-4-yl)methylidene]-1H-indole-3-carbohydrazide), has shown potent AChE and BChE inhibition, strong antioxidant activity, and low cytotoxicity in SH-SY5Y and Neuro-2a cells. Molecular docking studies suggest that 3a interacts with AChE, BChE, and melatonin MT1/MT2 receptors, supporting its potential as a neuroprotective agent. Angelova et al. (2023) explored two series of hybrid molecules combining melatonin and donepezil through hydrazone or sulfonyl hydrazone linkages. The lead indole-based hybrid compound 3a (N’-[(E)-(1-benzylpiperidin-4-yl)methylidene]-1H-indole-3-carbohydrazide) exhibited substantial AChE inhibition (76.51 ± 3.04 µM) and BChE inhibition (56.50 ± 0.20 µM), along with strong antioxidant and lipid peroxidation inhibition activities. In antioxidant assays, 3a showed robust 1,1-diphenyl-2-picrylhydrazyl (DPPH) activity and excelled in both ferric reducing antioxidant power (FRAP) and ferric thiocyanate (FTC) assays. Notably, 3a displayed low cytotoxicity in Neuro-2a (IC₅₀ > 300 µM) and SH-SY5Y cells (129 ± 7.5 µM), outperforming donepezil, and demonstrated favorable blood–brain barrier permeability (PAMPA BBB–logPe = 4.420). Molecular docking studies suggested that 3a targets AChE, BChE, and MT1 and MT2 receptors, marking it as a promising candidate for Alzheimer’s disease treatment (Angelova et al. 2023). In follow-up research (Mihaylova et al. 2024), the effects of 3a on Aβ-induced neurotoxicity and memory impairment were evaluated in mouse models. The N-benzylpiperidine derivative (3a) significantly affected Aβ₄₂ production in SH-SY5Y neuronal cultures after 24 h and showed superior anti-amyloid activity compared with the reference drugs DNPZ and galanthamine (GAL). Additionally, compound 3a administered alone reduced the amount of malondialdehyde (MDA) in mouse brain homogenate statistically significantly by 21.4%, while scopolamine (SC) increased it by 76% compared with the control group in the SC-induced brain toxicity test (Mihaylova et al. 2024). In animals treated with both SC and the test compound 3a, there was a statistically significant decrease in the MDA level by 19.3% compared with the group treated with SC alone. In contrast to DNPZ, compound 3a was able to protect cells in the dorsal hippocampus against SC-induced neurotoxicity (Mihaylova et al. 2024). Furthermore, 3a effectively influenced brain injury and AD-like pathology in the SC-induced dementia model in mice by attenuating cholinergic damage, lipid peroxidation (LP), and neuronal loss in the hippocampus (Mihaylova et al. 2024). Acute toxicity studies in mice (Mihaylova et al. 2024) showed that the compound is mildly toxic (500–5000 mg/kg) when administered orally and moderately toxic (50–500 mg/kg) when given intraperitoneally, based on the Hodge and Sterner scale (Government of Canada 2024). The low oral toxicity might be linked to poor oral bioavailability, as the compound may have limited absorption in the gastrointestinal tract or undergo significant hepatic and intestinal metabolism, yielding nontoxic metabolites. Further pharmacokinetic studies are needed to confirm this by measuring metabolite and parent compound levels in plasma, urine, and feces. Biochemical analysis of 3a revealed only slight differences from the reference drug donepezil, specifically in total protein and ASAT activity, indicating no associated hepatotoxic or nephrotoxic effects (Mihaylova et al. 2024). Intranasal drug delivery provides a unique opportunity for noninvasive access to the central nervous system. Its advantages include fast absorption, higher patient acceptability, and circumvention of metabolic degradation (Gandhi et al. 2024; Koo et al. 2024). Incorporation of active molecules into nanocarrier systems, particularly solid lipid nanoparticles, enhances their stability and bioavailability while offering controlled release and prolonged residence time in the nasal cavity (Costa et al. 2021; Zheng et al. 2024). These combined benefits make intranasal nanocarriers attractive candidates for the management of Alzheimer’s disease and related conditions (Nguyen and Duong 2025). Among the different nanocarrier platforms explored for nose-to-brain delivery, solid lipid nanoparticles (SLNs) have gained special attention due to their biocompatibility and potential to improve drug permeation. SLNs are colloidal systems that can deliver both hydrophilic and lipophilic active substances. Their core is a composite of solid lipids stabilized by a monolayer coating of surfactant on the surface (Schwarz et al. 1994). Their small particle size, as well as the presence of emulsifiers, determines their ability to pass through biological membranes and enables the partitioning of nanosized droplets in the nasal tissues, resulting in enhanced CNS delivery via intranasal application (Rizvi et al. 2019; Mehnert and Mäder 2001). Pharmacia 72: 1–13 3 The physiological lipids in their composition enhance the oral bioavailability of poorly water-soluble active substances (Mehnert and Mäder 2001), whereas their occlusive effect and excellent adhesion to mucous membranes increase nasal retention time (Kaur et al. 2008). Moreover, SLNs provide excellent drug loading, protection of the loaded active substance from degradation, improved stability, and superior controlled drug release. They are also biodegradable and have good tolerability, as there are no reports of acute or chronic toxicity, which makes them suitable for production scalability (Kaur et al. 2008; Helgason et al. 2009; Kumar et al. 2013). Considering these advantages, encapsulation of 3a into SLNs is expected to enhance stability, cellular uptake, and eventual brain delivery via the intranasal route. The present study aimed to prepare and characterize 3a-loaded SLNs, optimize their physicochemical properties, and evaluate their neuroprotective effects against H₂O₂-induced oxidative stress in SH-SY5Y neuronal cells. The in vitro model allows assessment of antioxidant and neuroprotective efficacy, providing insight into the potential of SLN-encapsulated 3a as a novel therapeutic strategy for AD. Materials and methods Stearic acid, palmitic acid, glyceryl monostearate, and glyceryl palmitostearate were purchased from Sasol Germany GmbH (Witten, Germany). Tween 80 was purchased from Sigma-Aldrich (St. Louis, USA). Ethanol, dimethyl sulfoxide (DMSO), disodium hydrogen phosphate dihydrate, and potassium dihydrogen phosphate were all purchased from Merck (Darmstadt, Germany). Deionized water was prepared in the laboratory. All solvents, chemicals, and reagents were obtained commercially and used without further purification. Synthesis and characterization of 3a A solution of 20 mmol of the corresponding carbonyl compound in 10 mL of absolute ethanol was mixed with a hot solution of 20 mmol aroylhydrazide in 10 mL of absolute ethanol at 60 °C and stirred for 1–8 h. The obtained crystalline precipitates were filtered, washed with an ethanol–ether mixture, and recrystallized from ethanol. After the chemical reactions, the crude compounds were purified through standard methods such as column chromatography and recrystallization to isolate the pure hybrid molecules. The final synthesized compounds were characterized by techniques such as nuclear magnetic resonance (NMR) spectroscopy, mass spectrometry, and elemental analysis to confirm their structures and purity. Development of an analytical method for 3a The assay of 3a was determined using UV–vis spectrophotometry. The method was validated with respect to selectivity, linearity, and precision. Evaluation of selectivity was performed by comparing the UV–vis spectra of the pure active pharmaceutical ingredient (API) and the placebo mixture in the range of 200–600 nm. Linearity of the analytical method was tested in two media: distilled water and pH 6.5 buffer. 3a (2 mg) was quantitatively transferred into 50 mL volumetric flasks and solubilized in 5 mL of DMSO, followed by dilution up to 50 mL with the respective medium. Seven standard stock solutions were diluted to cover the concentration range of 0.002–0.04 mg/mL. The tests were performed in triplicate. Calibration curves for the UV–vis spectrophotometrically measured stock solutions were plotted based on concentration versus absorbance. The correlation coefficient (R²) served as an indicator of linearity. Based on the calibration curve equation, the correlation coefficient, y-intercept, slope of the regression line, and residual sum of squares were determined and used in the formula for calculating the amount of API released from the dosage form. The precision was assessed by testing three concentration levels covering the specified range (50%, 100%, and 125% of the API concentration used in the standard experiments), preparing triplicates at each level. The tests were performed in both distilled water and pH 6.5 buffer media. Based on the obtained results, the relative standard deviation (RSD%) was calculated. Determination of solubility of 3a in solid lipids The solubility of 3a in stearic acid, palmitic acid, glyceryl monostearate, and glyceryl palmitostearate was evaluated semi-quantitatively. 3a was added in steps of 1 mg to 100 mg of melted lipid and stirred with a magnetic stirrer. The experiment continued until incomplete dissolution of the last added portion was observed. The test was repeated with the determined amount of 3a that showed complete dissolution. Solubility was evaluated visually in the melted lipid and microscopically in a thin section of the cooled solution (Shahraeini et al. 2020; Wolska and Brach 2022). Preparation of SLNs and loading of 3a The SLNs were prepared using the nano-template engineering technique with some modifications (Sohail et al. 2023). Glyceryl monostearate was melted at 70 °C. Tween 80 was homogenized in 10 mL of distilled water and then heated to 70 °C. The surfactant solution was added dropwise to the melted lipid under continuous stirring on a magnetic stirrer. After adding the last portion, stirring was continued at 800 rpm for 30 min. The obtained emulsion was sonicated for 4 min at 75% amplitude in a 20/10 s on/off cycle using a probe sonicator (Bandelin Sonopuls) to obtain a nanoemulsion. The sonicated nanoemulsion was placed into cold water (4 °C) to form the SLN dispersion. To prepare loaded SLNs, 10 mg of 3a was previously dissolved in the lipid. The amounts of lipid and surfactant for the different samples are presented in Table 2. The resulting dispersion was passed through a 0.45 µm syringe filter to eliminate unentrapped 3a and larger aggregates. Voycheva C et al.: SLNs loaded with a novel indole-based multitarget ligand4 SLNs were obtained after lyophilization at −80 °C for 24 h under a 1 × 10–4 mbar vacuum using an Alpha 3–4 LSCbasic semi-industrial freeze-dryer (Martin Christ Gefriertrocknungsanlagen GmbH, Osterode, Germany). The lyophilized SLNs were reconstituted with deionized water or buffer before use in further studies. Determination of SLN encapsulation efficiency (EE) The SLN (10 mL) suspension was centrifuged at 1300 rpm for 20 min. The supernatant, containing free nonencapsulated 3a, was suitably diluted with 10% DMSO and analyzed by UV spectrophotometry at λ = 258 nm, as described in the Development of an analytical method for 3a section. EE was calculated using the following equation: Dynamic light scattering (DLS) analysis The nanoparticle size, polydispersity index, and zeta potential were determined using a Zetasizer (Zetasizer Nano ZS, Malvern Panalytical, Worcestershire, UK). The samples (0.1% w/v) were dispersed in distilled water, sonicated for 20 min, and measured at a scattering angle of 90° and at 25 °C. Transmission electron microscopy (TEM) analysis The size and structure of 3a-loaded SLNs were characterized using transmission electron microscopy (JEOL JEM 2100 h STEM; 200 kV; point resolution = 0.23 nm). Samples were prepared by placing the aqueous suspension of nanoparticles on a polymer microgrid supported on a Cu grid. The water was subsequently evaporated under vacuum. X-ray diffraction analysis (XRD) X-ray powder diffraction patterns of the drug, lipid, and optimized 3a-SLNs were measured using an X-ray diffractometer (Bruker D8 ADVANCE, Germany). The samples were analyzed using Cu Kα radiation (45 kV, 40 mA) and scanned from 5° to 80° (2θ) with a step size of 0.03° (2θ) and a counting time of 17.5 s/step. Differential scanning calorimetry (DSC) DSC curves of 3a, SLNs, and 3a-SLNs were recorded using a differential scanning calorimeter (PerkinElmer DSC8500) equipped with an Intracooler 3 cooling system. The samples were scanned in the temperature range of −50 °C to 180 °C at a heating rate of 10 °C/min. Cooling was performed at 20 °C/min from 180 °C to 20 °C. Instrument control, data collection, and processing were conducted using Pyris software version 10.1.0.0412. In vitro drug release study The drug release from 3a-SLNs was evaluated in simulated nasal fluid (SNF; pH 6.5) using the dialysis bag method. The SNF medium contained 7.45 mg/mL NaCl, 1.29 mg/ mL KCl, and 0.32 mg/mL CaCl₂·2H₂O (Farid et al. 2013). To maintain sink conditions for 3a, 10% DMSO was added to the release medium. The dialysis membrane (pore size 2.4 nm; molecular weight cutoff 12,000–14,000; Sigma-Aldrich–MilliporeSigma) was soaked in distilled water for 12 h before use. The 3a-SLN dispersion, equivalent to 1.8 mg of 3a, was poured into the bag, and both ends were secured with clamps. The dialysis bag was placed in a 25 mL conical flask containing the dissolution medium. The release study was performed using an incubator shaker at 37 °C. Aliquots of the dissolution medium were withdrawn at predetermined time intervals, and an equal volume of fresh medium was added to maintain a constant volume. The samples were analyzed spectrophotometrically at 258 nm against a solvent blank. All experiments were performed in triplicate. In vitro permeability of 3a Permeability studies were performed using a Franz diffusion cell system (Logan Instruments Corp. 913-6 automated transdermal diffusion cell sampling system, Somerset, NJ, USA). The upper chamber (donor compartment) was filled with 2 mL of 3a-SLN dispersion equivalent to 1.8 mg of pure 3a (Zhang et al. 2020). The PermeaPad® membrane was placed between the donor and receptor compartments, with an effective surface area of 1.54 cm². The receptor compartment contained 12 mL of SNF (pH 6.5) with 10% DMSO added to maintain sink conditions. The stirring rate was maintained at 100 rpm, and the temperature was kept at 34 ± 0.5 °C using a circulating water bath throughout the 12 h permeation study (Nair et al. 2022). At predetermined intervals (1, 2, 3, 4, 5, 6, and 12 h), 1 mL of the sample was withdrawn from the receptor phase and replaced with fresh medium. The amount of permeated drug was analyzed spectrophotometrically at λ = 258 nm. All samples were tested in triplicate. The cumulative amount of the drug that penetrated through the membrane surface (Qt, µg/cm2) was calculated using the following equation (Liu et al. 2018): Qt = (Cn·VA + ∑i n-1 Ci ·Vs)/A where Qt is the cumulative amount of 3a that penetrated per unit membrane surface area; Cn is the 3a concentration in the n-th sample; Ci is the 3a concentration in the i-th sample; VA is the volume of the receptor phase (12 mL); Vs. is the volume of the withdrawn sample (2 mL); and A is the effective permeation area of the membrane (1.54 cm²). The cumulative Qt was plotted against time, and the steady-state flux (Jss, µg/cm²/h) was calculated from the slope of the linear portion of the curve (Liu et al. 2018). Pharmacia 72: 1–13 5 Stability studies The optimized 3a-SLN dispersion was subjected to stability testing in triplicate. Storage conditions were as follows: 4 ± 2 °C (refrigerated), 25 ± 2 °C and 60 ± 5% RH, and 40 ± 2 °C and 75 ± 5% RH for 6 months. The average particle size, polydispersity index, and zeta potential were measured under these conditions (Luo et al. 2006). Cell line and culture conditions The human neuroblastoma cell line SH-SY5Y (ECACC No. 94030304) was obtained from the European Collection of Authenticated Cell Cultures (ECACC, UK). This cell line is widely used as an in vitro model for neuronal differentiation, neurotoxicity, and neuroprotection due to its catecholaminergic properties and ability to acquire neuron-like characteristics under specific conditions. Cells were cultured in RPMI 1640 medium supplemented with 10% heat-inactivated fetal bovine serum (FBS), 2 mM L-glutamine, and 1% penicillin–streptomycin. Cultures were maintained at 37 °C in a humidified incubator with 5% CO₂. The medium was replaced every 2–3 days, and cells were subcultured at 70–80% confluence using trypsin–EDTA and reseeded at suitable densities for experiments. H₂O₂-induced oxidative stress model in SH-SY5Y neuronal cells To evaluate the cytoprotective potential of the test compound, both free 3a (0.1, 1, 5, 10, 25, and 50 µM) and nanoparticle-loaded 3a (3a-SLNs) at the same concentrations were tested. Unloaded nanoparticles were also assessed separately over a concentration range of 0.208–104 µg/mL to determine any effects of the carrier system alone. SHSY5Y cells were seeded in 96-well plates at 3.5 × 10⁴ cells per well and allowed to attach for 24 h under standard culture conditions (37 °C, 5% CO₂). Cells were then pretreated with the respective formulations for 90 min to allow cellular uptake. Following pretreatment, cells were washed with sterile PBS and exposed to 1 mM H₂O₂ for 10 min to induce oxidative stress. After the H₂O₂ challenge, fresh complete RPMI 1640 medium was added, and cells were incubated for an additional 24 h. Cell viability was subsequently measured using the MTT assay, with untreated cells serving as the negative control (100% viability) and H₂O₂-treated cells without compound or nanoparticles serving as the positive control for maximal oxidative damage. All data were analyzed using GraphPad Prism 8, applying one-way ANOVA followed by Dunnett’s post hoc test to determine statistically significant differences relative to the control groups. Results and discussion Synthesis and characterization of 3a The synthesis of 3a began with commercially available starting materials, such as indole derivatives and donepezil analogues, as presented in our previously published article (Angelova et al. 2023). A stepwise synthetic approach was used to incorporate indole and donepezil-like pharmacophores into a single hybrid molecule. Various chemical reactions, including nucleophilic substitution and condensation, were employed to form the desired hybrid structure. The synthesis aimed to optimize the balance between the two pharmacophores (indole and donepezil) to ensure that the final compound exhibited both acetylcholinesterase (AChE) inhibitory activity (donepezil-like) and affinity for other potential Alzheimer’s-related targets. After the chemical reactions, the crude compounds were purified through standard methods such as column chromatography and recrystallization to isolate the pure hybrid molecules. The final synthesized compounds were characterized by nuclear magnetic resonance (NMR) spectroscopy, mass spectrometry, and elemental analysis to confirm their structures and purity. The obtained compound was N’-[(E)- (1-benzylpiperidin-4-yl)methylidene]-2-(1H-indol-3-yl) acetohydrazide, and its structure is shown in Fig. 1. Development of an analytical method for 3a Validation of analytical methods is an essential process because it demonstrates that the analytical procedure used for a specific determination is suitable for its intended purpose and ensures its reliability by evaluating accuracy, precision, specificity, linearity, range, and robustness. Considering the assay of 3a from solid lipid nanoparticles, the method was validated with respect to selectivity, linearity, and precision. Selectivity refers to the extent to which a method can determine a particular analyte in a complex mixture without interference from other components (Valcárcel et al. 2001). To demonstrate selectivity, the UV–vis spectra of pure 3a and a placebo mixture containing glyceryl monostearate (GMS) and Tween 80 were recorded. The active pharmaceutical ingredient (API) exhibited a maximum absorption peak at 258 nm (λmax), whereas the placebo solution did not show any absorption at the same wavelength, confirming the selectivity of the assay method. Figure 1. Structure of N’-[(E)-(1-benzylpiperidin-4-yl)methylidene]-1H-indole-3-carbohydrazide (3a). Voycheva C et al.: SLNs loaded with a novel indole-based multitarget ligand6 According to ICH guideline Q2, the linearity of an analytical procedure is its ability (within a given range) to provide analytical test results that are directly proportional to the concentration (amount) of the API in the sample. Based on the UV–vis absorption determinations at 258 nm for the prepared standard stock solutions, calibration curves were constructed in distilled water and buffer medium (pH 6.5) (Figs 2, 3), and the corresponding equations were plotted. The high R² values confirmed the linear dependence between the concentration of 3a and its absorbance in the given concentration range in both media. The precision of an analytical procedure expresses the closeness of the results (degree of scatter) among a series of measurements obtained from multiple samplings of the same source under identical conditions. Precision data are generally expressed as standard deviation, relative standard deviation (RSD), and confidence interval. The RSD% results for 3a are shown in Fig. 4. RSD% values below 2% confirmed the precision of the analytical method for the assay of 3a. Solubility of 3a in lipids Initially, the solubility of 3a was determined in various lipids, and the results are presented in Table 1. Solubility studies indicated that 3a showed the highest solubility in glyceryl monostearate (GMS). Its selection for the present study was based on its solubilizing potential, biocompatibility, and suitability for nose-to-brain delivery (Joshi et al. 2012; Singh et al. 2012). The choice of surfactant was based on the hydrophilic– lipophilic balance (HLB) value. Tween 80, with an HLB of 15, can emulsify GMS to form a stable nanoemulsion at an acceptable concentration. Polysorbates are a suitable choice as coating materials in the preparation of carriers for brain-targeted delivery of active substances, as they facilitate transport across the blood–brain barrier (BBB) (Kreuter et al. 2003). Their mechanism of action is associated with endocytosis by brain capillary endothelial cells (Olivier 2005; Yadav et al. 2018). Several reports have shown that Tween 80 produces fine-sized SLNs suitable for nose-to-brain drug delivery (Gaur et al. 2014). After the selection of components, optimization was performed. The appropriate ratio of lipid and surfactant was chosen based on particle size, polydispersity index, zeta potential, and loading efficiency, as these parameters can influence in vivo permeability, toxicity, and brain targeting. Particle size has a significant effect on stability, drug loading, and release behavior, whereas particle size distribution affects circulation time, absorption, and biodistribution. Thus, small particle sizes and large surface areas lead to improved drug solubility, better mucoadhesion, and enhanced in vivo permeation compared with a pure drug solution or suspension. The results of the dynamic light scattering (DLS) analysis and encapsulation efficiency (EE%) calculations are presented in Table 2. In agreement with literature data, an increase in lipid concentration from 200 to 400 mg led to an increase in the particle size and PDI of SLNs (Shahraeini et al. 2020; Vitorino et al. 2011). The insufficient amount of Table 1. Solubility of 3a in lipids. Lipid Amount of lipid required* (mg) stearic acid 369.3 ± 1.52 palmitic acid 486.6 ± 2.64 glyceryl monostearate 219.7 ± 0.56 glyceryl palmitostearate 306.6 ± 1.14 *Data are expressed as mean ± SD, n = 3. Figure 2. Standard curve of 3a in distilled water with 10% DMSO (mean ± SD; n = 3). Figure 3. Standard curve of 3a in buffer (pH 6.5) with 10% DMSO (mean ± SD; n = 3). Figure 4. RSD% values for the five samples from three different concentrations of 3a, corresponding to 100%, 50%, and 125% of the test sample amount in distilled water and buffer (pH 6.5). Pharmacia 72: 1–13 7 surfactant required to emulsify GMS was the probable reason for this proportional dependency. The average particle size and PDI decreased with increasing Tween 80 content from 100 to 300 mg. This observation matches the findings of other authors who reported that the size of SLNs decreases with increasing surfactant concentration (Kumar et al. 2018). During sonication, the size of the droplets is reduced and a new surface is generated. An increase in surfactant concentration ensures stabilization of this newly formed surface. The polydispersity index (PDI) indicates the particle size distribution, with values below 0.3 confirming homogeneity and uniformity (Abousamra and Mohsen 2016). In the present study, PDI values below 0.3 were observed in samples with the lowest lipid and highest surfactant concentrations for both empty and loaded particles. The zeta potentials of the empty and loaded SLNs were comparable, likely due to their similar surface structures and the formation of a surfactant layer around the lipid matrix. The values ranged from −16.30 to −25.30 mV. The negative charge was probably induced by the adsorption of OH⁻ ions from water onto the SLN surface. Similar observations have been reported by other researchers (Ho and Ahmad 1999; Hsu and Nacu 2003; Abousamra and Mohsen 2016). Samples with a ζ-potential greater than 25 mV, particularly those with higher surfactant concentrations, exhibited better long-term colloidal stability for both empty and loaded SLNs. The highest EE% was observed in samples containing the highest lipid concentration. Since a lower lipid concentration leads to smaller particle size, drug molecules tend to escape from the lipid matrix, resulting in lower EE% (Singh et al. 2012). Partial expulsion of the active substance can occur at lower lipid concentrations during crystallization of the lipid phase. EE% also decreased with decreasing surfactant concentration, likely due to particle coalescence caused by insufficient Tween 80. This was confirmed by the increased PDI values observed when surfactant concentration was reduced in loaded particles. SLNs with smaller mean particle sizes can effectively cross the BBB, whereas those with a PDI below 0.3 and a zeta potential above −25 mV are expected to exhibit the greatest long-term stability. Based on these findings, samples 3a/1 (empty) and 3a-SLN/1 (loaded) were selected for further investigation. Transmission electron microscopy (TEM) investigation The TEM image presented in Fig. 5 shows the spherical shape and homogeneous shading of the nanoparticles loaded with 3a. The particle size, ranging from approximately 50 to 60 nm, as confirmed by TEM, is consistent with the Zetasizer measurements. Differential scanning calorimetry (DSC) The DSC thermogram of 3a showed a sharp endothermic melting peak in the temperature range of 150–160 °C, with a maximum at 158 °C (Fig. 6). The peak of SLNs appeared between 50 °C and 80 °C, with a maximum at 75 °C. The 3a-SLNs peak exhibited higher intensity and a broader range than that of the empty SLNs due to the incorporation of 3a within the lipid crystal lattice. The thermogram of 3a-SLNs did not show the melting peak of 3a, suggesting that 3a was dissolved in the lipid matrix and existed in an amorphous state. X-ray diffractometric analysis In the X-ray diffraction (XRD) pattern of 3a, sharp peaks at 2θ-scattering angles of 24.2°, 33.6°, 34.8°, and 35.9° were distinctly noticeable, indicating its high crystallinity (Fig. 7). The crystalline nature of the empty SLNs was evident from peaks at approximately 20.6°, 24.0°, Table 2. Samples, formulation, and physicochemical properties of empty and 3a-loaded SLNs (mean ± SD; n = 3). S № 3a (mg) GMS (mg) Tween 80 (mg) PS (nm) PDI Zp (mV) EE % 3a/1 – 200 300 47.96 ± 2.02 0.288 –25.30 ± 4.06 – 3a/2 – 300 300 63.67 ± 4.03 0.368 –23.23 ± 2.02 – 3a/3 – 400 300 99.17 ± 1.36 0.513 –21.18 ± 3.03 – 3a/4 – 200 100 78.16 ± 4.12 0.415 –16.30 ± 0.32 – 3a/5 – 200 200 59.87 ± 4.01 0.313 –20.23 ± 3.21 – 3a-SLN/1 10 200 300 56.23 ± 4.36 0.268 –25.16 ± 0.17 89.5 ± 3.65 3a-SLN/2 10 300 300 80.44 ± 0.02 0.467 –22.12 ± 2.11 92.6 ± 1.73 3a-SLN/3 10 400 300 103.24 ± 3.51 0.543 –21.11 ± 0.42 95.1 ± 2.34 3a-SLN/4 10 200 100 98.10 ± 6.87 0.473 –17.89 ± 4.27 83.6 ± 0.45 3a-SLN/5 10 200 200 78.56 ± 2.09 0.389 –21.25 ± 1.16 86.4 ± 3.21 S – Sample, GMS – glyceryl monostearate, PS – particle size, PDI – polydispersity index, Zp – zeta potential, EE% – entrapment efficiency. Figure 5. TEM image of 3a-SLNs (sample 3a-SLN/1). Voycheva C et al.: SLNs loaded with a novel indole-based multitarget ligand8 25.3°, and 25.6°. In the spectra of 3a-SLNs, the characteristic peaks of 3a were absent, and the lipid peaks displayed lower intensity. This observation suggests that 3a was successfully incorporated into the SLNs. The reduced peak intensity of 3a-SLNs was probably due to the intercalation of 3a within the lipid crystal lattice, which altered the crystallinity of the loaded SLNs. In vitro drug release study In vitro release of 3a from 3a-SLNs was compared with the diffusion of free 3a across the dialysis membrane (Fig. 8). The study was conducted in simulated nasal fluid (SNF; pH 6.5). Nearly 99% of free 3a crossed the dialysis barrier within 3 h. In contrast, about 38% of 3a was released from 3a-SLNs during the first 6 h of the dissolution test, followed by sustained release reaching 52% at 24 h. The initial burst release was likely due to the presence of 3a near the SLN surface, whereas the subsequent sustained release resulted from the extended diffusion path through the lipid matrix. Similar release profiles have been reported for SLNs prepared by the nano-template engineering technique (Qureshi et al. 2016, 2017; Rizvi et al. 2019; Sohail et al. 2023). In vitro permeability assay In vitro permeability assays are essential in drug research and development. Most permeability studies simulating intranasal delivery employ ex vivo animal membrane models, such as sheep nasal mucosa, due to their structural similarity to human mucosa (Sharma et al. 2014). However, these standard ex vivo models often exhibit poor reproducibility and variability in results across tissue sources, laboratories, and researchers (Hayeshi et al. 2008). Moreover, the use of animal membranes raises ethical concerns and is time-consuming, as tissues must be incubated under specific conditions before testing (Di Cagno et al. 2015S). To obtain more reproducible results, the present study employed PermeaPad®, a non-cellular, phospholipid vesicle-based permeation assay (PVPA) method, instead of ex vivo models (Ma and Wu 2017; Rahamim and Azagury 2021). PermeaPad® is a biomimetic membrane that simulates passive diffusion across various biological barriers (gastrointestinal, buccal, and nasal) and provides a more reliable assay because its barrier consists of phosphatidylcholine liposomes (Lecithin S-100) immobilized between nitrocellulose filter supports, mimicking the structure and geometry of biological membranes (Cagno and Bauer-Brandl 2019). Due to this unique design, the PermeaPad® barrier is robust, cost-effective, easy to use (without pretreatment), and resistant to a wide pH range (1–10) as well as to surfactants and co-solvents such as DMSO (Bibi et al. 2015). The Franz cell diffusion system was used to analyze the permeation of 3a from 3a-SLNs (sample 3a-SLN/1), and the diffusion was compared with that of free 3a (3a dispersion). The study was conducted in SNF (pH 6.5), corresponding to the nasal environment, for 12 h. The permeation profiles shown in Fig. 9 illustrate the differences in 3a behavior between the two formulations. The results Figure 6. DSC analysis of 3a, SLNs, and 3a-SLNs. Figure 7. X-ray diffractometric analysis of 3a, SLNs, and 3a-SLNs. Figure 8. In vitro release of 3a from 3a dispersion and 3a-SLNs (sample 3a-SLN/1) in pH 6.5 at 34 °C (mean ± SD; n = 3). Pharmacia 72: 1–13 9 clearly demonstrated that drug transport from 3a-SLNs was significantly higher (1.27-fold) than from free 3a. This enhancement could be attributed to the intrinsic lipid structure of the SLNs (resembling biological lipids), their small size, and the presence of emulsifiers, which facilitate penetration through the PermeaPad® membrane due to its lipid composition similar to nasal mucosa. The findings suggest that the prepared 3a-SLNs could also promote the partitioning of nanosized droplets within the nasal mucosa, thereby increasing residence time and enhancing brain drug delivery following intranasal administration of 3a-SLNs. The slower release of compound 3a from solid lipid nanoparticles (SLNs) through a dialysis membrane compared with its faster passage through PermeaPad® can be attributed to several factors related to the material properties of the barriers and the SLN formulation. The dialysis membrane used for release studies is typically composed of a semipermeable material with a defined pore size (2.4 nm in this study) and molecular weight cutoff (12,000–14,000 Da). This membrane primarily allows passive diffusion of free drug molecules but can restrict the passage of drug-loaded nanoparticles or larger aggregates, resulting in slower drug release. In contrast, PermeaPad® is a phospholipid-based biomimetic membrane designed to simulate biological membranes. Its lipidic composition closely resembles that of cellular membranes, enhancing the interaction and diffusion of lipid-based carriers such as SLNs. The SLN structure also plays a significant role in the permeation process. In SLNs, the drug (3a) is encapsulated within a lipid matrix. Drug release occurs in two stages—an initial burst release of surface-associated drug followed by sustained diffusion through the lipid core. The lipid matrix provides a controlled release environment, reducing the rate at which 3a becomes available for diffusion through the dialysis membrane. The sustained release behavior of 3a-SLNs observed through the dialysis membrane is consistent with the extended diffusion path that the drug must traverse within the lipid matrix before reaching the release medium. In addition, PermeaPad®, due to its lipidic nature, may facilitate interaction with SLNs, enhancing the partitioning and transport of drug-loaded nanoparticles or released drug. Conversely, the dialysis membrane lacks this lipid compatibility, which may reduce interaction and permeation of both SLNs and the released drug. Particle size and surface characteristics are additional factors contributing to higher permeability. The small size (50–60 nm) and lipid-based composition of SLNs improve their interaction with lipidic membranes such as PermeaPad®. These characteristics may promote better partitioning into the PermeaPad® membrane, enhancing the rate of drug release and transport. In conclusion, the differences in the release profiles can be explained by the contrasting mechanisms of interaction and transport through the respective membranes, with PermeaPad® offering a more efficient model for simulating biological membrane interactions. Stability studies No significant changes were observed in particle size during storage at 4 ± 2 °C (refrigerated) or at room temperature (25 ± 2 °C, 60 ± 5% RH). However, particle size increased significantly (p < 0.05) under accelerated conditions (40 ± 2 °C, 75 ± 5% RH) due to aggregation (Table 3). Zeta potential and polydispersity index did not show significant changes when the nanoparticle dispersion was stored at 4 ± 2 °C and 25 ± 2 °C, 60 ± 5% RH. At 40 ± 2 °C and 75 ± 5% RH, the PDI increased. A possible explanation for this observation is the change in the zeta potential value from −25 mV in the initial month to −15 mV in the third month and −6 mV in the sixth month of storage under these conditions. This variation might be due to partial dissolution of the lipid coating, which leads to particle aggregation. H₂O₂-induced oxidative stress model in SH-SY5Y neuronal cells To evaluate the neuroprotective potential of the newly synthesized compound 3a, SH-SY5Y cells were pretreated with various concentrations (0.1–50 µM) of free 3a or 3a-SLNs (sample 3a-SLN/1) for 90 min, followed by exposure to 1 mM H₂O₂ to induce oxidative stress. Exposure to H₂O₂ for 15 min significantly reduced cell viability compared with untreated controls (Fig. 10). Pretreatment with free 3a provided only modest protection, significant at 25 and 50 µM, preserving 9% and 16% of cell viability, respectively (p < 0.05; p < 0.001 vs. H₂O₂). In contrast, 3a-SLNs exhibited a concentration-dependent neuroprotective effect over a broader range (1–50 µM), preserving cell viability by 7%, 9%, 17%, 26%, and 36%, respectively (p < 0.05; p < 0.01; p < 0.001 vs. H₂O₂), and demonstrating significantly stronger protection than the free compound Figure 9. In vitro permeability of 3a from 3a dispersion and 3a-SLNs (sample 3a-SLN/1) across PermeaPad® membrane in SNF (pH 6.5) for 12 h (mean ± SD; n = 3).