Synergistic Neuroprotection in Parkinson's Disease via Photobiomodulation and Liposomal Rosmarinic Acid Delivery

Abstract

Parkinson's Disease (PD) is a progressive neurodegenerative disorder characterized by dopaminergic neuronal loss, oxidative stress, and mitochondrial dysfunction. Current treatment strategies are largely symptomatic and fail to halt disease progression. This research work explores a novel dual-modal therapeutic strategy combining Photobiomodulation (PBM) using near-infrared (NIR) light with nanotechnology-enhanced delivery of Rosmarinic Acid (RA) for the treatment of PD. Building upon the findings of previous works, which established the neuroprotective potential of RA, this study extends its application to PD treatment through the development of RA-loaded liposomes (RA@LP) and their integration with NIR-induced PBM. As a noninvasive modality, NIR light has demonstrated efficacy in stimulating mitochondrial activity, promoting ATP production, and reducing oxidative stress through PBM mechanisms. In parallel, RA, a potent natural antioxidant, has been encapsulated within liposomal nanocarriers to enhance its stability, bioavailability, and targeted delivery to affected neuronal tissues. The combined therapeutic platform of PBM and RA@LP is designed to eliminate endogenous and exogenous reactive oxygen species (ROS), thereby breaking the self-perpetuating cycle of oxidative stress and mitochondrial damage underlying PD pathogenesis. We highlight in vitro investigations that demonstrate the synergistic effects of PBM and RA@LP on neuronal cells. The results indicate that this dual approach protects mitochondrial integrity and improves cellular viability under PD-like oxidative conditions. By broadening the scope to include in vitro analysis, the study provides deeper mechanistic insights into the cellular responses to light-based and nanomedicine therapies. This work presents a promising, noninvasive, and multitargeted strategy for PD treatment, with potential implications for translational research. Integrating phototherapy and nanotechnology represents a significant advancement in developing effective neuroprotective interventions.

Keywords: Parkinson’s disease; and reactive oxygen species; liposome; photobiomodulation; rosmarinic acid.

1

NIR PBM activates cytochrome c oxidase in mitochondria, boosting ATP production and stabilizing mitochondrial function, thereby protecting cells from oxidative damage. Meanwhile, RA@LP reduces oxidative stress by scavenging reactive oxygen species, chelating metal ions, and enhancing antioxidant enzymes.

2

Characterization of RA@LP. TEM image of (a) LP and (b) RA@LP. (c) Size distribution, (d) ζ-potential, and (e) UV–vis absorption of LP and RA@LP. (f) Calibration curve for RA quantification. (g) The emission spectra of RA at various concentrations in ethanol were recorded using an excitation wavelength of 328 nm. Stability test results of (h) LP and (i) RA@LP in deionized water and PBS show their behavior under different aqueous environments.

3

Biocompatibility assay of LP and RA@LP formulations. (a) Photograph of hemolysis test tubes and (b) hemolysis assay of LP and RA@LP. (c) The schematic diagram of the Transwell system design mimicking the BBB. The FITC-tracking BBB penetration results of (d) fluorescence intensity and (e) confocal microscopy (*p < 0.05, **p < 0.01, ***p < 0.001 compared to the control group).

4

(a) Line graph illustrating the encapsulation efficiency (EE) and loading concentration of RA@LP at various RA concentrations shows the relationship between the input RA concentration and the resulting encapsulation performance. (b) ROS remaining ratio (inset: color change diagram of DPPH solution after reaction with the drug). (c) The stability assay was based on DPPH scavenging (*p < 0.05, **p < 0.01, ***p < 0.001 compared to the control group). (d) The rotenone-induced PD models. (e) The differentiation scheme of SH-SY5Y cells and (f) observing the cell morphology under the optical microscope.

5

Cell viability assays of RA and RA@LP treated (a) normal SH-SY5Y cells and (b) ROT-induced PD cell model. Cell viability assays of NIR treated (c) normal SH-SY5Y cells and (d) PD model, where normal cell is denoted as NC. (e) Cell viability assays of the PD model with the RA@LP and PBM. (f) Bliss synergy score of NIR and RA@LP. (g) Scatter plot and regression curve of luminescence versus log­[ATP] for the ATP standard solution. (h) ATP level assays of the PD model with the RA@LP and PBM. (i) Bliss synergy score of NIR and RA@LP. (The significance is marked as *p < 0.05, **p < 0.01, and ***p < 0.001 compared to the control group. The significance is marked as #p < 0.05, ##p < 0.01, and ###p < 0.001 compared to the ROT group).

6

(a) Western blot image and (b) quantification histogram of ATP synthase expression. Superoxide dismutase (SOD) activity analysis with (c) scatter plot and regression curve of absorbance values versus time for the SOD standard solution, (d) scatter plot and regression curve of % inhibition versus the logarithm of concentration for the SOD standard solution, (e) scatter plot and regression curve of absorbance values versus time for samples under different treatment methods, and (f) bar graph of SOD concentrations and their relative proportions across different treatment groups. Bliss synergy score of (g) ATP5C1 expression and (h) SOD activity.

7

DCF assay exhibited in (a) fluorescence intensity and (d) confocal microscopy. The JC-1 assay is shown in (b) fluorescence intensity and (e) confocal microscopy. Bliss synergy score of (c) membrane potential. The significance is marked as *p < 0.05, **p < 0.01, and ***p < 0.001 compared to the control group. The significance is marked as #p < 0.05, ##p < 0.01, and ###p < 0.001 compared to the ROT group.