Formulation, Development and Evaluation of NDDS Formulation for Treatment of Parkinson’s Disease

Vaishali Parag Rawal¹*, Dr. Subodh Anil Gangurde²

1.    Research Scholar, School of Pharmaceutical Sciences, Sandip University, Mahiravani, Nashik -422213, Maharashtra, India

2.    Associate Professor, School of Pharmaceutical Sciences, Sandip University, Mahiravani, Nashik-422213, Maharashtra, India

*Correspondence: vaishu.rawal@gmail.com

DOI: https://doi.org/10.71431/IJRPAS.2025.4705

INTRODUCTION

Parkinson’s disease is a widespread and progressive neurological disorder that impacts over 10 million people globally. [1] It is marked by both motor and non-motor symptoms. [2]The motor-related issues—such as resting tremors, slowness of movement (bradykinesia), and muscle stiffness—are primarily due to the deterioration of dopaminergic neurons in the nigrostriatal pathway.[3]However, many individuals experience non-motor symptoms like sleep disturbances, depression, and cognitive changes well before motor signs appear. [4]These early issues suggest that neurodegeneration begins in brain areas beyond the dopaminergic system, highlighting a broader pathological process. Only recently has modern medicine come to understand that these early non-motor symptoms may signal the onset of Parkinson’s disease itself. [5]

The aim of the study is to construct, stable easily administer NDDS formulation with improved bioavailability and reduction in side effect. Data of formulation of Nanoliposomes, nanocochleates and animal study in rat model Parkinson’s disease induced by 6-OHDA has been already been communicated for publication [6, 7] the study also involve evaluation of formulation by cell line study. This research article gives the details of cell line study.

Cell line studies have become indispensable in modern biomedical research, offering a controlled and reproducible platform to investigate cellular physiology, disease mechanisms, and therapeutic interventions. A cell line refers to a population of cells derived from a single source that can proliferate indefinitely under in vitro conditions. These models provide a consistent and scalable alternative to primary cells and animal models, enabling researchers to conduct experiments with high precision and reproducibility. [8, 9]

Over the past decades, cell lines have played a pivotal role in advancing our understanding of cancer biology, virology, immunology, and pharmacology. Their utility spans from drug screening and toxicity testing to genetic manipulation and vaccine development. Moreover, the emergence of immortalized and genetically engineered cell lines has further expanded the scope of experimental possibilities, allowing for targeted studies on specific cellular pathways and disease phenotypes. [10]

Despite their widespread use, cell line studies are not without limitations. Issues such as cross-contamination, genetic drift, and phenotypic alterations over time necessitate rigorous authentication and quality control. Nonetheless, when properly validated and maintained, cell lines remain a cornerstone of translational research, bridging the gap between basic science and clinical application. [10, 11]

This study aims to explore cell viability when cytotoxic effect is induced by H2O2 on human epithelial cell lines" to the growing body of knowledge that supports the development of effective and safe therapeutic strategies.

Material and Method

A) Cell line, Reagents and kits

SH-SY5Y cell line was obtain by the National centre for cell science (NCCS) Pune, India. Dulbecco's Modified Eagle's medium (DMEM), foetal bovine serum (FBS), and phosphate buffer saline were purchase from Invitrogen (Carlsbad, USA). All other chemicals and reagents used in this study were of analytical grade. Enzyme-linked immunosorbent assay (ELISA) kits were purchase from Ray Biotech Inc, USA.

·         Cell culture and culture conditions

SH-SY5Y cells were cultured in DMEM supplemented with 10% heat-inactivated FBS, 100 µg/ml streptomycin and 50 units/ml penicillin. The cells were incubated at 37°C in the presence of 5% CO₂ and sub-cultured every 2 days. [8]

·         Cell viability assay Protects against H2O2-Induced Cytotoxicity

Analysis of Cell Viability: Cell viability was determined by the MTT (3-(4,5- dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay. SH-SY5Y cells were seeded in 96-well plates at a density of 1 * 10⁴ cell/well and incubated for 24 h prior to experimental treatments. The cells were then subjected to the samples. After 24-h incubation, MTT (0.5 mg/ml) was added to each well. Following an additional 3-h incubation at 37°C, 100 µl of DMSO was added to dissolve the formazan crystals. The absorbance was then measured at 540 nm using an ELISA plate reader (Bio-Rad Laboratories, CA, USA). Wells without cells were used as blanks and were subtracted as background from each sample. Results were expressed as a percentage of control.[12]                                                                                                           

Lactate Dehydrogenase (LDH) Release Assay:

Cells dying by apoptosis or necrosis released LDH into the supernatant. The amount of LDH in the supernatant was measured with a cytotoxicity detection kit (Roche). In brief, the cells (1 * 10⁴ cell/well) were seeded in 96-well plates and then treated with H₂O₂ for indicated periods after being pretreated with or without samples for 1 h. For analysis, 100 µl supernatant was extracted from each well and was placed in separate wells of a new 96-well plate, and 100 µl catalyst solutions was added to each well and incubated at 37°C for 30 min. Absorbance was measured at 490 nm using an ELISA plate reader (Bio-Rad Laboratories, CA, USA). Total cellular LDH was determined by lysing the cells with 2% Triton X-100 (high control) the assay medium served as a low control and was subtracted from all absorbance measurement. Measure the absorbance at 490nm and 680nm. LDH activity is determined by subtracting the680nm absorbance value (background) from the 490nm absorbance before calculation of %Cytotoxicity [(LDH at 490nm) - (LDH at 680nm)].[13,14,15,16]

To calculate % Cytotoxicity, subtract the LDH activity of the Control LDH Release (water treated) from the Activator/inhibitor-treated sample LDH activity, divide by the total LDH activity

[(Total LDH Release activity) – (Control LDH Release activity)], and multiply by 100                             

Results and discussion

SH-SY5Y cells were treated with different concentrations of levodopa and its two formulations (20-100mcg/ml) for12 h and the cell viability was determined by MTT assay. When exposed to LDNC of 100 mcg/ml or lower, the viability of SH-SY5Ycells was (85.09%) nearly the same as untreated control cells (87.77%) (Fig 1, Table-1 – MTT assay@24 hrs)

Fig 1- MTT assay @24hrs

In order to evaluate whether H₂O₂ influences neuronal cytotoxicity, SH-SY5Y cells were treated with various concentrations of H₂O₂ (0, 1, 10, 50, 150, and 300 µM) for12 h. Cytotoxicity in SH-SY5Y cells is H₂O₂ induced a dose-dependent. (Fig 2)

In order to determine the protective effects of levodopa and its formulations against H₂O₂-induced loss of cell viability, SH-SY5Ycells were pretreated with 10 µg/ml samples respectively for 1 h, followed by treatment with 150 µM H₂O₂ for 12 h.H₂O₂-induced loss of cell viability was significantly attenuated by LDNC than LDNL and LD. (Fig 3)

In order to further investigate the protective effect of LD, LDNL, LDNC, the release of LDH was measured. LDH release is increased as the number of dead cells increases. The release of LDH was   increased significantly after exposure to 150 µM H₂O₂, indicating that H₂O₂ caused cytotoxicity inSH-SY5Y cells. In contrast, the samples showed decreasing release of LDH from least to maximum by LD, LDNL, LDNC compared with H₂O₂-exposed cell group. The protective effect of samples on H₂O₂-induced cytotoxicity determined by LDH assay was similar to that determined by MTT assay. The highest viability of cells against the neurotoxicity induced by H₂O₂was attained by LDNC and then LDNL and LD, suggesting the protective effect of levodopa is better in its nanocochleates form.

CONCLUSION

The present in vitro study effectively demonstrated the protective potential of Levodopa and its nanoformulations against oxidative stress-induced cytotoxicity in SH-SY5Y neuronal cells, a well-established model for Parkinson’s disease. Exposure to hydrogen peroxide (H₂O₂) significantly reduced cell viability and increased LDH release, confirming its role as a potent cytotoxic agent.

Among the tested formulations, Levodopa nanocochleates exhibited superior neuroprotective efficacy, as evidenced by significantly higher cell viability in the MTT assay and reduced LDH leakage, compared to free Levodopa and Levodopa-loaded liposomes. These findings suggest that nanocochleate-based delivery enhances cellular uptake, stability, and sustained release of Levodopa, thereby mitigating oxidative damage more effectively.

Overall, this study supports the potential of Levodopa nanocochleates as a promising drug delivery system for improving therapeutic outcomes in Parkinson’s disease, warranting further in vivo validation and mechanistic investigations.

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