Evaluation of the neuroprotective effects of alpha lipoic acid-loaded folate-conjugated chitosan nanoparticles against 6-OHDA-induced apoptosis and oxidative stress in an in vitro Parkinson’s disease model

Folate receptors, which mediate the cellular uptake of folic acid (FA) for essential processes such as DNA synthesis and repair, are expressed on neurons affected in Parkinson’s disease (PD). While the etiology of PD remains incompletely understood, oxidative stress is implicated as a key contributor. Alpha-lipoic acid (ALA) is a potent antioxidant; however, its therapeutic application is limited by instability, low bioavailability, and an unpleasant odor. Nanotechnology offers a promising strategy to overcome these limitations. This study aimed to develop and characterize folic acid-conjugated chitosan nanoparticles encapsulating ALA (FA-CS-ALA NPs) and to evaluate their efficacy against 6-hydroxydopamine (6-OHDA)-induced neurotoxicity. The FA-CS-ALA NPs, characterized by transmission electron microscopy, exhibited an irregular spherical morphology. Dynamic light scattering (DLS) analysis determined an average particle size of 658.13 nm and a polydispersity index (PDI) of 0.17, indicating moderate size distribution. In vitro studies using SH-SY5Y neuroblastoma cells demonstrated that 6-OHDA exposure significantly increased oxidative stress, neuroinflammation, and apoptosis. Both free ALA and FA-CS-ALA NPs effectively mitigated these deleterious effects. Notably, the FA-CS-ALA NPs exhibited superior neuroprotective efficacy compared to free ALA, suggesting that the folate-conjugated nanocarrier enhances therapeutic delivery.

Graphical Abstract

Introduction

Parkinson’s disease (PD) is a complex neurodegenerative disorder characterized by diverse clinical manifestations and a multifactorial etiology. As the second most prevalent neurodegenerative condition globally, the number of PD cases is projected to double over the next three decades, underscoring a pressing public health challenge [1, 2]. While the degeneration of dopaminergic neurons in the substantia nigra pars compacta and the resulting striatal dopamine deficiency underpin the cardinal motor symptoms, the precise pathogenesis remains incompletely understood. Accumulating evidence implicates oxidative stress as a pivotal mechanism, with the unique metabolic microenvironment of the substantia nigra predisposing it to cytotoxic free radical generation and subsequent neuronal damage [3, 4]. Current PD management is primarily symptomatic. Levodopa remains the first-line treatment for motor disability, and optimal care often requires a multidisciplinary approach incorporating non-pharmacological interventions. However, no available therapy has been proven to slow or halt disease progression, highlighting a critical unmet need in PD therapeutics [5, 6]. This limitation is compounded by a long prodromal phase, during which significant neurodegeneration occurs prior to clinical diagnosis. The development of disease-modifying strategies, particularly those capable of targeted neuroprotection, is therefore a paramount objective. A promising therapeutic candidate for addressing oxidative stress in PD is (R)-(+)-lipoic acid (RLA), the biologically active enantiomer of alpha-lipoic acid (ALA). RLA is a potent, endogenous antioxidant and an essential cofactor for mitochondrial enzymes. Its therapeutic potential, however, is constrained by physicochemical limitations, including instability, low bioavailability, and non-specific tissue distribution [7, 8]. To overcome these challenges, nanoparticle-based drug delivery systems offer a compelling solution. Such systems can enhance the stability and bioavailability of therapeutic compounds while enabling targeted delivery [9]. Among various nanocarriers, chitosan a polysaccharide derived from the N-deacetylation of chitin has emerged as an excellent material due to its biocompatibility, biodegradability, and versatile formulation properties [10]. Furthermore, chitosan itself has demonstrated intrinsic antioxidant activity [11, 12]. To achieve neuronal targeting, we functionalized chitosan nanoparticles with folic acid. This strategy capitalizes on the high expression of folate receptors on neurons, potentially enhancing the specificity and cellular uptake of the delivered therapeutic agent [13–15]. In this study, we investigated the neuroprotective efficacy of alpha-lipoic acid encapsulated within folate-conjugated chitosan nanoparticles (FA-CS-ALA NPs) against 6-hydroxydopamine (6-OHDA) induced neurotoxicity. The neurotoxin 6-OHDA is widely used to model PD in vitro, as it selectively damages the nigrostriatal pathway, inducing oxidative stress, neuroinflammation, and apoptotic neuronal death that recapitulate key features of the disease [16, 17]. Using the SH-SY5Y neuroblastoma cell line, we aimed to evaluate whether the FA-CS-ALA NP formulation provides superior protection compared to free ALA, thereby validating the potential of this targeted delivery system for Parkinson’s disease intervention.

Materials and methods

Cell line and reagents

Medium-molecular weight (MMW) Chitosan (CS ; 190–310 kDa ), Folic acid (97%, FA), 1-Ethyl-3-(3 dimethyl aminopropyl) carbodiimide (EDC), N-hydroxy succinimide (NHS), acetic acid, trypsin-EDTA (0.25%), 6-hydroxydopamine (purity ≥ 97%, 6-OHDA), and MTT were procured from the Sigma Aldrich company. Sodium tripolyphosphate (TPP) from Samchun Chemical Company of Korea was also used. Alpha-lipoic acid (purity > 97%, ALA) were provided by Gol Exir Pars (Iran). Fetal Balf Serum (FBS), and DMEM/F12 were provided by Gibco (UK). Dimethyl sulfoxide (DMSO) and Ethanol (96%, v/v) were provided by Mojallali Co. (Iran). The Cellular reactive oxygen species (ROS) Assay Kit was prepared Abcam (UK). Additionally, The annexin V/PI kit came from Zist Pajouhan Mahbob in Tehran. The SH-SY5Y neuroblastoma cell line was obtained from Iran’s Pasteur Institute (Tehran, Iran). It is a suitable choice for this investigation due to its extensive prior use in related studies [18, 19]. Cells were maintained in DMEM/F12 media, supplemented with FBS (10%,v/v), and penicillin/streptomycin (1%,v/v). Cells were incubated at 37 °C with 5% CO₂ in a humidified incubator.

Preparation of FA-CS (Folic acid-conjugated chitosan)

Folate-conjugated chitosan was synthesized using carbodiimide chemistry following established protocols [20]. Briefly, 200 mg of CS was dissolved in 100 mL of 1% (v/v) acetic acid solution. Separately, a mixture containing 17.5 mg EDC, 8 mg NHS, and 200 mg folic acid was prepared by dissolving the components in 10 mL anhydrous DMSO under constant stirring at room temperature (RT) until complete dissolution. This solution was then added to the CS solution and stirred at RT in the dark overnight. Subsequently, the pH was adjusted to 9 using 1 M NaOH. The precipitate was collected by centrifugation, washed thoroughly, and the resulting yellow product was freeze-dried for future use.

Preparation of ALA-Loaded FA-CS nanoparticles (FA-Cs-ALA NPs )

FA-CS-ALA nanoparticles were synthesized using an ionic gelation technique with modifications [21]. First, 20 mg of the synthesized FA-CS conjugate was dissolved in 20 mL of 1% (v/v) acetic acid under magnetic stirring in the dark for 16 h at RT. Separately, 10 mg of ALA was dissolved in 2 mL of DMSO. The ALA solution was then added to the FA-CS solution, and the pH was adjusted to 4.8 using 1 M NaOH, followed by 20 min of stirring. Nanoparticle formation was induced by adding 10 mL of an aqueous 0.5% (w/v) TPP solution dropwise into 22 mL of the FA-CS/ALA mixture under constant magnetic stirring. Stirring was continued for 1 h to allow complete nanoparticle formation. The resulting FA-CS-ALA NPs were collected by centrifugation at 7000×g for 15 min and lyophilized.

Nanoparticle characterization

The successful conjugation of folic acid to chitosan was confirmed by comparing the ultraviolet (UV) absorption spectra and Fourier-Transform Infrared (FTIR) spectra of FA, CS, and the FA-CS conjugate. The morphology of the FA-CS-ALA NPs was examined using transmission electron microscopy (TEM, LEO910, Germany). The hydrodynamic diameter, polydispersity index (PDI), and zeta potential of the nanoparticles were determined by Dynamic light scattering (DLS) and electrophoretic light scattering using a Horiba SZ-100 analyzer. FTIR spectra were recorded on a Nicolet Avatar spectrometer (USA) in the range of 400–4000 cm−¹ to identify functional groups and confirm ALA encapsulation. The crystalline structure of the formulations was assessed by X-ray diffraction (XRD; Philips PW1730) using Cu Kα radiation (λ = 0.154 nm) over a 2θ range of 10–80°.

Determination of loading capacity (LC) and encapsulation efficiency (EE)

To calculate the percentage of loading capacity (LC) and encapsulation efficiency (EE), the calibration curve of ALA was determined by UV-Vis spectroscopy at 337 nm. After the preparation of ALA-loaded FA-CS nanoparticles at the final step, the supernatant was separated and the amount of unloaded ALA was obtained through a calibration curve (y = 1340.9x-30.727, R = 0.99). Then, LC and EE % were calculated by following the formula:

1

2

In vitro drug release study

The drug release profile of ALA from FA-CS-ALA NPs was evaluated according to a published method [22]. Briefly, 10 mg of FA-CS-ALA NPs were suspended in 10 mL of phosphate-buffered saline (PBS, pH 7.4) and incubated at 37 °C with continuous shaking (100 rpm). At predetermined time intervals (0, 1, 3, 5, 18, 24, 48, 72, and 96 h), 1 mL of the release medium was withdrawn and replaced with an equal volume of fresh pre-warmed PBS to maintain sink conditions. The concentration of released ALA in the samples was quantified by measuring the absorbance at 337 nm using a UV-Vis spectrophotometer. The cumulative percentage of ALA release was plotted as a function of time. The release kinetics were analyzed by fitting the data to various mathematical models using the DDSolver program [23]. For comparative mechanistic analysis, a reference dissolution profile for free ALA was simulated [24, 25]. Given the high solubility of ALA in the dissolution medium and its inherently rapid dissolution kinetics as an unformulated powder, this simulated profile was designed to represent an instantaneous release, reaching > 95% within 5 h and plateauing at 100% thereafter. This simulated profile of free ALA serves as a baseline control to underscore the release-retarding efficacy of the developed nanoparticle formulation.

Cell viability assessment

Determination of IC₅₀

The cytotoxicity of 6-OHDA, free ALA, and FA-CS-ALA NPs was determined using the MTT assay [26]. SH-SY5Y cells (1 × 10⁴ cells/well) were seeded in 96-well plates and cultured for 24 h. Cells were then treated with: 6-OHDA (1–1000 µM), ALA (1–8000 µg/mL), and FA-CS-ALA NPs (1–1000 µg/mL). After 24 h incubation, media was replaced with 100 µL fresh medium containing MTT (0.5 mg/mL) for 3 h. The formazan crystals formed were dissolved in 100 µL of DMSO, and the absorbance was measured at 570 nm using a Stat Fax 2100 microplate reader (Awareness Technology, USA). The half-maximal inhibitory concentration (IC₅₀) was calculated from the dose-response curves. Untreated cells served as the control.

Neuroprotective evaluation

In 96-well plates, SH-SY5Y cells (1 × 10⁴ cells/well) were divided into four groups (n = 3).Control group: Culture media only, ALA pretreatment: Non-toxic ALA doses (7.5 and 15 µg/mL, 24 h) followed by 125 µM 6-OHDA (24 h), FA-CS-ALA NPs pretreatment: NPs (7.5 and 15 µg/mL, 24 h) followed by 125 µM 6-OHDA (24 h), and 6-OHDA group: 125 µM 6-OHDA (½ IC₅₀) alone. Cell viability was quantified using the MTT assay as described above.

Apoptosis analysis by flow cytometry

The extent of apoptosis induced by 6-OHDA and the protective effects of the treatments were evaluated using an Annexin V-FITC/PI staining kit according to the manufacturer’s instructions [27]. SH-SY5Y cells were seeded in 12-well plates at a density of 3 × 10⁵ cells/well. After 24 h, cells were pretreated with ALA or FA-CS-ALA NPs (7.5 and 15 µg/mL) for 24 h, followed by exposure to 125 µM 6-OHDA for a further 24 h. The cells were then trypsinized, washed with cold PBS, and resuspended in 500 µL of 1X binding buffer. Subsequently, cells were stained with 2 µL of Annexin V-FITC for 10 min in the dark, followed by the addition of 1 µL of propidium iodide (PI). Apoptotic cells were quantified using a BD Biosciences flow cytometer, and data were analyzed using FlowJo software (v7.6.1, Tree Star).

Measurement of intracellular ROS

Intracellular ROS levels were measured using the fluorescent probe 2’,7’-dichlorofluorescin diacetate (DCFDA). SH-SY5Y cells were seeded in 96-well black plates at a density of 2.5 × 10⁴ cells/well. After 24 h, the cells were loaded with 10 µM DCFDA in serum-free medium and incubated for 45 min in the dark. Following incubation, the DCFDA solution was removed, and the cells were washed with PBS. The cells were then treated with ALA or FA-CS-ALA NPs (7.5 and 15 µg/mL) for 1 h prior to the addition of 125 µM 6-OHDA for 4 h. The fluorescence intensity was measured at excitation/emission wavelengths of 485/535 nm using a fluorescence microplate reader (Becton Dickinson, USA). Untreated cells served as the negative control.

Statistical analysis

All experiments were performed in at least triplicate. Data are presented as the mean ± standard deviation (SD). Statistical analysis was performed using GraphPad Prism software (v8, USA). The normality of the data distribution was confirmed using the Shapiro-Wilk test. Differences between multiple groups were analyzed by one-way analysis of variance (ANOVA), followed by an appropriate post-hoc test. A p-value of less than 0.05 was considered statistically significant.

Results

Characterization of the FA-CS conjugate

UV-Vis spectroscopy

The UV-Vis spectra of CS, FA, and FA-CS were recorded to confirm FA conjugation on CS (Fig. 1). The results showed FA has two absorption peaks at 299 and 374 nm and CS has a peak at 300 nm which are assigned to n-π* and π- π* transitions. The FA-CS showed two peaks at 287 and 374 nm which is in accordance with UV-Vis spectra of FA and CS and confirms FA connected to the CS. In addition, high yield absorption of FA-CS may indicate high purity of FA-CS.

Fig. 1.

The UV-Vis spectra of FA, CS, and FA-CS

FTIR spectroscopy

The FTIR spectra of FA-CS, CS, and FA were measured in the range of 400 to 4000 cm^− 1 to investigate the interaction between FA and CS (Fig. 2). The FTIR spectra of FA-CS and CS showed similar bands. The wide band at 3446 cm^− 1 is related to hydroxyl groups. The bands at 2885,1388, and 1087 cm^− 1 are scribed to the stretching vibration of C-H, bending vibration of CH₂, and vibration of C-O-C, respectively. The characteristics benzene ring bands appeared at 1654 and 1595 cm^− 1 which related to C = O, N-H. However, the intensity of the bands at these regions shifted in the FTIR spectrum of FA-CS. Also, the band at 1654 cm^− 1 disappeared and also the band at 1595 cm^− 1 sharpened and shifted to 1579 cm^− 1 which indicates the formation bond between amide of CS and carboxyl of FA. Therefore, the results of FTIR spectra are in agreement with UV-Vis results and confirm the successful conjugation of FA and forming the FA-CS.

Fig. 2.

The FTIR spectra of FA, CS, and FA-CS

DLS and zeta potential analysis

The successful conjugation was further verified by changes in the hydrodynamic diameter and surface charge of the nanoparticles (Fig. 3). Blank CS nanoparticles had an average size of 274.3 nm, a PDI of 0.16, and a zeta potential of + 31.38 mV. Following FA conjugation, the FA-CS nanoparticles exhibited a larger hydrodynamic diameter of 600.64 nm (PDI = 0.2) and a significantly reduced zeta potential of + 4.96 mV. This substantial decrease in positive charge is consistent with the conjugation of FA to the primary amine groups on the CS backbone, which neutralizes the surface charge [28, 29]. Moreover, the PDI value in both nanoparticles was favorable and the size of the nanoparticles was at the nanoscale.

Fig. 3.

The DLS and zeta potential results of CS (a) and FA-CS nanoparticles (b)

Characterization of FA-CS-ALA nanoparticles

The morphology and particle size of FA-CS-ALA nanoparticles were determined by TEM image (Fig. 4), where indicated nanoparticles are spherical shape and have smooth edges with an average size of 49.29 ± 7.4 nm. Additionally, the FESEM images show that nanoparticles are spherical and confirm TEM results. The size and morphology of nanoparticles are critical factors for drug delivery in which sizes less than 10 nm would quickly eliminate from renal filtration and also, reticuloendothelial system and non-specific scavenging by monocytes can recognize nanoparticles and eliminate them. Also, the type of chitosan and crosslinker affect the size of nanoparticles [30–32], Therefore, these nanoparticles are suitable for tumor drug delivery. The DLS and zeta potential analysis of FA-CS-ALA and FA-CS nanoparticles were employed for hydrodynamic diameter and stability. The result of DLS nanoparticles shows that FA-CS-ALA is 658.13 nm with PDI of 0.17 and the zeta potential of particles was still − 15.7 mV even after a month (Fig. 5). As a comparative table of key parameters represented in Table 1, despite the size of nanoparticles got larger after folate conjugation and drug encapsulation, they were still at the nanoscale and favorable size. Also, the PDI in all of them was suitable and indicates nanoparticles are monodispersed. The zeta potential of CS was like other reports and after folate conjugation, the zeta was reduced due to interaction with amine groups. Then, the zeta potential of FA-CS-ALA changed to a negative charge which is because hydroxyl groups mostly outwarded after encapsulation [33–35]. The XRD and FTIR analyses were recorded to confirm drug encapsulation. The XRD patterns of FA-CS and FA-CS-ALA are presented in Fig. 6a. The FA-CS is an amorphous structure due to network formation between polymers and crosslinker and three sharp peaks appeared at 12, 20, and 50°. As XRD pattern of FA-CS-ALA exhibited, after ALA encapsulation, the crystalline structure of nanoparticles decreased and peaks slightly shifted which might be due existence of ALA molecules in polymeric nanoparticles [36]. The FTIR spectrum of FA-CS-ALA in Fig. 6b shows bands at regions of 3463, 2925, 1392, 1101, and 553 cm^{− 1} which are scribed to OH, Ch, CH2 and C-O-C vibration of CS, are shifted to higher wavenumber after ALA encapsulation. Also, the band at 898 cm^{− 1} appeared and the band at 1604 cm^{− 1} was sharpened after encapsulation which might be due to the interaction of CS with hydroxyl groups of ALA and overlapping carboxylic acid groups of ALA with amine groups of CS, respectively. These results suggest that hydroxyl and carbonyl groups of ALA are mostly responsible for ALA and FA-CS interaction [37].

Fig. 4.

The TEM image of FA-CS nanoparticles (a), particle size histogram (b), and FESEM images at zoom scales 200 (c) and 500 nm (d)

Fig. 5.

The DLS and zeta potential results of FA-CS-ALA nanoparticles (a) and after a month (b)

Table 1.

A comparison of key parameters of nanoparticles

Sample	Size (nm)	Zeta (mV)	PDI
CS	274.35	38.32	0.16
FA-CS	600.64	4.96	0.25
FA-CS-ALA	658.13	-18.26	0.17
FA-CS-ALA (after a month)	1778	-15.7	0.57

Fig. 6.

The XRD patterns of FA-CS and FA-CS-ALA (a) and FTIR spectra of ALA, FA-CS, and FA-CS-ALA nanoparticles (b)

Encapsulation efficiency and loading capacity

The encapsulation efficiency and loading capacity are critical factors for drug delivery vehicles that indicate the amount of drug that is successfully entrapped into chitosan nanoparticles and the amount of drug loaded per unit of nanoparticles [38]. The EE was calculated at 93.6% through Eq. 2, which determines the high encapsulation efficiency of ALA. The LC was calculated at 78% by Eq. 1, which also indicates a high loading capacity. These values indicate that the ionic gelation method is highly effective for entrapping ALA within the chitosan-based nanoparticles.

In vitro release profile of ALA

The release kinetics of ALA from FA-CS-ALA NPs were evaluated in PBS (pH 7.4) at 37 °C. As shown in Fig. 7, the nanoparticles demonstrated a biphasic release pattern: an initial burst release (~ 30% within 6 h) followed by sustained release over 96 h, ultimately achieving 80% cumulative release by day 4. This controlled release profile suggests stable encapsulation and gradual diffusion of ALA under physiological conditions. Furthermore, the release kinetics were evaluated based on mathematical models (Table 2) by fitting the release data. The release profile of free ALA showed an excellent fit to the First-Order model, with an R² value of 0.99 and a release rate constant of 0.95 h−¹. These parameters indicate rapid, concentration-dependent dissolution, and the high solubility of ALA confirms that ALA does not have inherent sustained-release properties [39].In contrast, the encapsulated formulation showed the best fit to the Korsmeyer-Peppas model (R²= 0.94) with a release exponent of n = 0.30 [40].This result demonstrates a Fickian diffusion-controlled release mechanism, indicating that the nanoparticles successfully modulated drug release by creating a polymeric barrier through which the drug must diffuse, rather than allowing instantane ous release.

Fig. 7.

Release profiles of ALA from FA-CS-ALA nanoparticles compared to the simulated release profile of the free drug in phosphate buffer pH = 7.4. The free drug profile was simulated based on the intrinsic high solubility and rapid dissolution characteristics of ALA

Table 2.

Kinetic model fitting parameters for ALA release

Model	R²_adj	AIC	Parameters
First-Order	0.99	40.2	k1 = 0.95
Higuchi	0.81	66.9	kH = 9.9
Korsmeyer-Peppas	0.94	57.5	k = 22, n = 0.3

Cytotoxicity assessment

The cytotoxicity of 6-OHDA, free ALA, and FA-CS-ALA NPs was evaluated using the MTT assay. Treatment with 6-OHDA induced dose-dependent cytotoxicity in SH-SY5Y cells, with an IC₅₀ value of 246.2 µM (Fig. 8a). Based on this, a sub-cytotoxic concentration of 125 µM (½ IC₅₀) was selected for subsequent neuroprotection studies. Both free ALA and FA-CS-ALA NPs showed concentration-dependent effects on cell viability (Fig. 8b, c). Non-toxic concentrations of 7.5 and 15 µg/mL for both ALA and FA-CS-ALA NPs were chosen for the neuroprotection experiments.

Fig. 8.

The effect of 6-OHDA (a), ALA (b) and, FA-CS-ALA NPs (c) on SH-SY5Y cells viability

Neuroprotective effects against 6-OHDA

Pre-treatment with FA-CS-ALA NPs (7.5 and 15 µg/mL) significantly attenuated 6-OHDA-induced cytotoxicity (p < 0.001 vs. 125 µM 6-OHDA group) (Fig. 9). The protective effect of the FA-CS-ALA NPs was significantly greater than that of free ALA at equivalent concentrations, demonstrating the advantage of the nanoformulation.

Fig. 9.

The protective effects of pre-treatment with ALA and FA-CS-ALA NPs against 125 µM 6-OHDA-induced cytotoxicity. Results are presented as the mean ± SD of three independent experiments. (**p < 0.001 as compared to the untreated control group. *p < 0.005 and ***p < 0.0001 as compared to the 6-OHDA-treated group)

Anti-apoptotic effects

Our findings demonstrated a significant increase in apoptotic cells after 6-OHDA treatment of SH-SY5Y cells at a concentration of 125 µM compared to the control (p < 0.001). Pre-treatment with ALA and FA-CS-ALA NPs significantly ameliorated these effects (from 59% of apoptotic cells in the 6-OHDA group to 49.4% and 35.0% in the 7.5 µg/mL ALA and 7.5 µg/mL FA-CS-ALA NPs pre-treated groups, respectively; p < 0.001) (Fig. 10).

Fig. 10.

The effect of ALA and FA-CS-ALA NPs on apoptosis rate caused by 6-OHDA in SH-SY5Y cells. (a) Flow cytometry analysis was used to evaluate the apoptosis in each group. (b) Quantification of apoptotic SH-SY5Y cell number in each group. The results are expressed as the mean ± SD oftriplicate tests. (*p < 0.005 as compared to the control group and as compared to 6-OHDA-treated group)

Reduction of intracellular ROS

Compared to the untreated control cells, 6-OHDA treatment of SH-SY5Y cells markedly increased the generation of ROS. Treatment with ALA alone considerably decreased intracellular levels of ROS compared to the control group. Furthermore, it became clear that pretreatment with FA-Cs-ALA remarkably reduced the amount of ROS caused by 6-OHDA compared to the group that received only 6-OHDA (Fig. 11).

Fig. 11.

The effect of ALA and FA-CS-ALA NPs on the intracellular ROS levels induced by 6-OHDA in SH-SY5Y cells. The results are presented as the mean ± SD of triplicate tests. (*p < 0.005 as compared to the untreated control group, and as compared to 6-OHDA-treated group)

Discussion

This study demonstrates that alpha-lipoic acid encapsulated within folate-conjugated chitosan nanoparticles (FA-CS-ALA NPs) confers superior neuroprotection against 6-OHDA-induced toxicity in SH-SY5Y cells compared to free ALA. Our findings are supported by comprehensive nanoparticle characterization and multiple biological assays, confirming the promise of this targeted delivery system for PD intervention. The successful synthesis of the FA-CS conjugate was verified by spectroscopic analyses, and the resulting FA-CS-ALA NPs exhibited favorable physicochemical properties, including a nanoscale size, moderate polydispersity, and negative surface charge, which contributed to their stability over one month. However, an increase in the mean hydrodynamic diameter of the nanoparticles from 658.13 to 1778 nm was observed. Although this indicates particle aggregation, the zeta potential only changed slightly from − 18.2 to -15.7 mV, demonstrating excellent surface charge stability. The combination of these findings suggests a reversible flocculation mechanism, whereby individual particles associate into larger clusters without losing their fundamental surface properties. The maintenance of a negative surface charge (-15.7 mV) provides a sufficient energy barrier to prevent irreversible aggregation and indicates good colloidal stability over the studied period. Furthermore, the formed aggregates were redispersible upon mild sonication, which is standard practice for many nanoparticulate formulations prior to administration [41, 42]. The high encapsulation efficiency and loading capacity further validate the ionic gelation method for effectively incorporating ALA. The in vitro release profile, characterized by an initial burst followed by sustained release, is ideal for achieving both immediate and prolonged therapeutic effects. From a mechanistic insight, the n-value of 0.3, which is lower than 0.45, clearly indicates Fickian diffusion [40]. This signifies that the drug release is primarily governed by the diffusion of ALA molecules through the aqueous pores of the hydrated polymer network. The stark contrast between the rapid first-order release of free ALA (k = 0.95 h−¹) and the slow, diffusion-controlled release from the encapsulated formulation underscores the efficacy of the encapsulation process. Furthermore, the superior fit of the Korsmeyer-Peppas model over the Higuchi model, as analyzed through standard model comparison practices [39], provides additional confirmation of the proposed diffusion-controlled mechanism.The core finding of this work is the significantly enhanced neuroprotective efficacy of the FA-CS-ALA NPs. Pre-treatment with the nanoformulation more effectively restored cell viability, reduced apoptosis, and attenuated intracellular ROS levels than equivalent doses of free ALA. This enhanced performance can be attributed to several factors inherent to the nanodelivery system. First, the conjugation of folic acid facilitates receptor-mediated endocytosis, leveraging the high expression of folate receptors on neuronal cells to improve cellular uptake and specificity [13, 14]. Second, the chitosan matrix itself may contribute synergistic benefits. Chitosan and its nanoparticles have been independently shown to possess intrinsic antioxidant properties and can ameliorate oxidative stress and inflammation in various injury models [43–47]. By encapsulating ALA, the system likely combines the direct antioxidant action of ALA with the bio-enhancing and protective properties of the chitosan carrier. The results align with the established therapeutic profile of ALA, a potent natural antioxidant known to induce Nrf2-mediated hormetic responses, upregulate cytoprotective enzymes like heme oxygenase-1 (HO-1), and suppress NF-κB-driven neuroinflammation [48–51]. However, the therapeutic potential of free ALA is often limited by its pharmacokinetic properties. Our study, consistent with other reports, confirms that nanoparticle encapsulation can overcome these limitations. For instance, the enhanced efficacy of drug-loaded chitosan nanoparticles compared to free drugs has been demonstrated in models of cardiotoxicity and malaria, where the nanoformulations improved bioavailability and targeted delivery [52, 53].

Limitations of the study

While the results are promising, several important limitations must be acknowledged:

1. Most notably, while we employed folic acid as a targeting ligand to potentially enhance delivery via folate receptor (FR)-mediated endocytosis, we did not provide direct experimental validation for receptor-specific cellular uptake.Supportive data from competitive inhibition assays or direct cellular uptake tracking would be required to conclusively demonstrate the targeting mechanism. Therefore, the enhanced efficacy of the FA-CS-ALA NPs, while significant, may be attributed to a combination of factors, including general nanoparticle effects and the proposed targeting. The definitive role of FR-mediated uptake remains a compelling question for future investigation.

2. The SH-SY5Y neuroblastoma cell line, while widely used, does not fully recapitulate the complexity of mature dopaminergic neurons in vivo. The relevance of folate receptor expression in this cell line to neurons in the human substantia nigra requires further validation.

3. As noted, the proposed mechanism of folate-receptor mediated targeting is inferred but not directly proven. Furthermore, the specific intracellular signaling pathways modulated by the FA-CS-ALA NPs were not investigated in depth.

4. The study would have been strengthened by including a control group of non-targeted CS-ALA NPs (without folate). This control is essential to definitively attribute the enhanced efficacy to folate-mediated targeting, as opposed to a general nanoparticle effect.

5. Although a control group with blank (drug-free) FA-CS nanoparticles was not included, the lack of cytotoxicity observed for the FA-CS-ALA NPs at the administered concentrations (Fig. 8b), coupled with the well-documented biocompatibility and intrinsic protective properties of chitosan [44–47], strongly suggests that the observed neuroprotection is primarily mediated by the delivered alpha-lipoic acid. Future studies will include this control to provide further definitive evidence.

6. The most significant limitation is the absence of in vivo data. The 6-OHDA model, while useful, does not present the blood-brain barrier (BBB), which is the major obstacle for CNS drug delivery. The ability of these nanoparticles to cross the BBB remains entirely unknown and is a critical area for future investigation [54].

Conclusion

In conclusion, we have successfully developed and characterized folate-conjugated chitosan nanoparticles for the targeted delivery of alpha-lipoic acid. The FA-CS-ALA NP formulation demonstrated significant advantages over free ALA, providing enhanced protection against 6-OHDA-induced oxidative stress, apoptosis, and neuroinflammation in an in vitro model of Parkinson’s disease. This enhanced efficacy is likely due to improved cellular uptake via folate-receptor mediated endocytosis and the synergistic effects of the chitosan carrier. This study underscores the potential of receptor-targeted nanocarriers as a strategic approach to improving the bioavailability and therapeutic outcome of neuroprotective agents. Future work will focus on optimizing the formulation for improved in vivo stability and BBB penetration, paving the way for developing effective nanotherapies for neurodegenerative diseases.

Abbreviations

FA

Folic acid

PD

Parkinson’s disease

ALA

Alpha-lipoic acid

FA-CS-ALA NPs

Folic acid-conjugated chitosan nanoparticles encapsulating ALA

6-OHDA

6-hydroxydopamine

DLS

Dynamic light scattering

PDI

Polydispersity index

RLA

(R)-(+)-lipoic acid

MMW

Medium-molecular weight

CS

Chitosan

EDC

1-Ethyl-3-(3 dimethyl aminopropyl) carbodiimide

NHS

N-hydroxy succinimide

TPP

Sodium tripolyphosphate

FBS

Fetal Balf Serum

DMSO

Dimethyl sulfoxide

ROS

Reactive oxygen species

RT

Room temperature

TEM

Transmission electron microscopy

UV

Ultraviolet

FTIR

Fourier-Transform Infrared

XRD

X-ray diffraction

LC

Loading capacity

EE

Encapsulation efficiency

IC₅₀

The half-maximal inhibitory concentration

DCFDA

The fluorescent probe 2’,7’-dichlorofluorescin diacetate

SD

Standard deviation

ANOVA

One-way analysis of variance

HO-1

Heme oxygenase-1

Author contributions

E.G. was responsible for the conception and design of the study, developed the synthesis strategy, synthesized and performed the majority of expriments presented in this study and drafted the manuscript. M.J. design the experimets, support technically, analyzed the data. P.H. was contributed in manuscript drafting. S.M. characterized the nanocarrier, and authored the manuscript. A.A. performed the expriments and analysed the data. M.S. provided valuable insights and experiences that significantly improved the quality of the research. All authors meticulously reviewed and endorsed the final manuscript, thereby guaranteeing its clarity and overall excellence.

Funding

This study is related to the project No. 4031206from Pharmacological Research Center of Medicinal Plants, Mashhad University of Medical Sciences, Mashhad, Iran.

Data availability

No datasets were generated or analysed during the current study.

Declarations

Ethics approval and consent to participate

Not applicable.

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.