Intranasal delivery of DPSC-derived small extracellular vesicles-encased phloroglucinol attenuates non-motor and motor deficits and promotes neurogenesis in an in vivo rat model of Parkinson’s disease

Abstract

Background  Parkinson’s disease (PD) is characterized by dopaminergic (DA) neuron degeneration in the substantia nigra pars compacta (SNpc) driven by oxidative stress, inflammation, and impaired neurogenesis. Phloroglucinol, a polyphenolic antioxidant, has demonstrated neuroprotective effects in PD models but suffers from limited clinical applicability due to poor blood-brain barrier (BBB) permeability. Small extracellular vesicles (sEV) derived from dental pulp stem cells (DPSCs) exhibit neuroprotective and immunomodulatory properties and serve as promising vehicles for targeted drug delivery across the BBB. This study aimed to evaluate the therapeutic efficacy of intranasally administered sEV-encased phloroglucinol (sEV-Phl) in a chronic MPTP rat model of PD. Methods  DPSC-derived sEV were isolated via density gradient ultracentrifugation and characterized using Transmission Electron Microscopy (TEM), Dynamic-Light-Scattering (DLS), and CD marker expression. Phloroglucinol was encased in sEV (sEV-Phl) using sonication. Antioxidant properties were tested in vitro using an H2DCF.DA assay in SH-SY5Y cells exposed to 6-OHDA. Chronic MPTP-treated male Wistar rats received intranasal sEV-Phl, with motor and non-motor behaviours evaluated up to 4-weeks post-MPTP treatment. TH-positive neurons, neurogenesis (Ki67, BrdU and FOXA2), lipid-peroxidation, and neurotransmitter-levels were analyzed. sEV biodistribution was tracked via near-infrared imaging and localization in neuronal and glial cells was confirmed with PKH-26 labelling, with confocal-imaging further verifying localization in neuronal and glial cells. TNF-α expression was assessed as a marker of neuroinflammation. Results  sEV displayed high purity and homogeneity. sEV-Phl significantly reduced oxidative stress both in vitro and in vivo, as indicated by decreased ROS and lipid peroxidation levels. sEV-Phl treated MPTP rats demonstrated marked improvement in motor and non-motor behaviours compared to MPTP rats. Immunohistochemical analysis revealed

1. Introduction

Parkinson’s disease (PD) is a complex neurodegenerative disorder marked by the selective loss of dopaminergic (DA) neurons in the substantia nigra pars compacta (SNpc) of the midbrain, resulting in striatal dopamine depletion and disruption of basal ganglia circuitry [1, 2]. By the time motor symptoms of PD manifest, an estimated 60–70% of midbrain DA neurons have already been lost [3, 4]. Despite significant advances in recent years, current pharmacological and other therapeutic interventions are yet not able to halt PD progression or support the survival of existing DA neurons. Dopamine and its analogues are the mainstay in PD therapy, but their long-term use is associated with progressively severe side-effects and the production of reactive oxygen species (ROS) due to autoxidation, DA neurons being particularly susceptible to oxidative damage due to the presence of both dopamine and high levels of iron.

While ROS (which contribute to oxidative stress) cannot be directly estimated in living patients or post-mortem tissues due to their short half-life, several indirect indicators of ROS activity in the post-mortem Parkinsonian brain support the significant role of oxidative stress in the disease. These include increased membrane peroxidation, as indicated by elevated levels of TBA (thiobarbituric acid)-reactive substances; increased cysteinyl adducts of dopamine; and ROS-mediated DNA damage, evidenced by higher levels of 8-hydroxy-2-deoxyguanosine in the substantia nigra pars compacta (SNpc) [5–7]. In addition to oxidative stress, inflammation and the lack of compensation for damaged/lost DA neurons are two other key factors that significantly contribute to the progression and worsening of neurodegeneration in PD. Unfortunately, dopamine analogues do not address these factors, and thus effective treatment in PD should incorporate supportive therapies that can provide antioxidative, anti-inflammatory and regenerative relief, ideally delivered through a non-invasive route.