Neuroprotective effects of intranasal extracellular vesicles from human platelet concentrates supernatants in traumatic brain injury and Parkinson's disease models.

The burgeoning field of regenerative medicine has significantly advanced with recent findings on biotherapies using human platelet lysates (HPLs), derived from clinical-grade platelet concentrates (PCs), for treating brain disorders. These developments have opened new translational research avenues to explore the neuroprotective effects of platelet-extracellular vesicles (PEVs). Their potential in managing neurodegenerative conditions like traumatic brain injury (TBI) and Parkinson's disease (PD) warrants further exploration. We aimed here to characterize the composition of a PEV preparation isolated from platelet concentrate (PC) supernatant, and determine its neuroprotective potential and neurorestorative effects in cellular and animal models of TBI and PD. We isolated PEVs from the supernatant of clinical-grade PC collected from healthy blood donors utilizing high-speed centrifugation. PEVs were characterized by biophysical, biochemical, microscopic, and LC-MS/MS proteomics methods to unveil biological functions. Their functionality was assessed in vitro using SH-SY5Y neuronal cells, LUHMES dopaminergic neurons, and BV-2 microglial cells, and in vivo by intranasal administration in a controlled cortical impact (CCI)-TBI model using 8-weeks-old male C57/BL6 mice, and in a PD model induced by MPTP in 5-month-old male C57/BL6 mice. PEVs varied in size from 50 to 350 nm, predominantly around 200 nm, with concentrations ranging between 1010 and 1011/mL. They expressed specific platelet membrane markers, exhibited a lipid bilayer by cryo-electron microscopy and, importantly, showed low expression of pro-coagulant phosphatidylserine. LC-MS/MS indicated a rich composition of trophic factors, including neurotrophins, anti-inflammatory agents, neurotransmitters, and antioxidants, unveiling their multifaceted biological functions. PEVs aided in the restoration of neuronal functions in SH-SY5Y cells and demonstrated remarkable neuroprotective capabilities against erastin-induced ferroptosis in dopaminergic neurons. In microglial cells, they promoted anti-inflammatory responses, particularly under inflammatory conditions. In vivo, intranasally delivered PEVs showed strong anti-inflammatory effects in a TBI mouse model and conserved tyrosine hydroxylase expression of dopaminergic neurons of the substantia nigra in a PD model, leading to improved motor function. The potential of PEV-based therapies in neuroprotection opens new therapeutic avenues for neurodegenerative disorders. The study advocates for clinical trials to establish the efficacy of PEV-based biotherapies in neuroregenerative medicine.

Delila L ，Nebie O ，Le NTN ，Timmerman K ，Lee DY ，Wu YW ，Chou ML ，Buée L ，Chou SY ，Blum D ，Devos D ，Burnouf T ... -  《-》

The neuroprotective activity of heat-treated human platelet lysate biomaterials manufactured from outdated pathogen-reduced (amotosalen/UVA) platelet concentrates.

Nebie O ，Devos D ，Vingtdeux V ，Barro L ，Devedjian JC ，Jonneaux A ，Chou ML ，Bordet R ，Buée L ，Knutson F ，Blum D ，Burnouf T ... -  《-》

Human platelet lysate biotherapy for traumatic brain injury: preclinical assessment.

Traumatic brain injury (TBI) leads to major brain anatomopathological damages underlined by neuroinflammation, oxidative stress and progressive neurodegeneration, ultimately leading to motor and cognitive deterioration. The multiple pathological events resulting from TBI can be addressed not by a single therapeutic approach, but rather by a synergistic biotherapy capable of activating a complementary set of signalling pathways and providing synergistic neuroprotective, anti-inflammatory, antioxidative, and neurorestorative activities. Human platelet lysate might fulfil these requirements as it is composed of a plethora of biomolecules readily accessible as a TBI biotherapy. In the present study, we tested the therapeutic potential of human platelet lysate using in vitro and in vivo models of TBI. We first prepared and characterized platelet lysate from clinical-grade human platelet concentrates. Platelets were pelletized, lysed by three freeze-thaw cycles, and centrifuged. The supernatant was purified by 56°C 30 min heat treatment and spun to obtain the heat-treated platelet pellet lysate that was characterized by ELISA and proteomic analyses. Two mouse models were used to investigate platelet lysate neuroprotective potential. The injury was induced by an in-house manual controlled scratching of the animals' cortex or by controlled cortical impact injury. The platelet lysate treatment was performed by topical application of 60 µl in the lesioned area, followed by daily 60 µl intranasal administration from Day 1 to 6 post-injury. Platelet lysate proteomics identified over 1000 proteins including growth factors, neurotrophins, and antioxidants. ELISA detected several neurotrophic and angiogenic factors at ∼1-50 ng/ml levels. We demonstrate, using two mouse models of TBI, that topical application and intranasal platelet lysate consistently improved mouse motor function in the beam and rotarod tests, mitigated cortical neuroinflammation, and oxidative stress in the injury area, as revealed by downregulation of pro-inflammatory genes and the reduction in reactive oxygen species levels. Moreover, platelet lysate treatment reduced the loss of cortical synaptic proteins. Unbiased proteomic analyses revealed that heat-treated platelet pellet lysate reversed several pathways promoted by both controlled cortical impact and cortical brain scratch and related to transport, postsynaptic density, mitochondria or lipid metabolism. The present data strongly support, for the first time, that human platelet lysate is a reliable and effective therapeutic source of neurorestorative factors. Therefore, brain administration of platelet lysate is a therapeutical strategy that deserves serious and urgent consideration for universal brain trauma treatment.

Nebie O ，Carvalho K ，Barro L ，Delila L ，Faivre E ，Renn TY ，Chou ML ，Wu YW ，Nyam-Erdene A ，Chou SY ，Buée L ，Hu CJ ，Peng CW ，Devos D ，Blum D ，Burnouf T ... -  《-》

In vitro evaluation of platelet extracellular vesicles (PEVs) for corneal endothelial regeneration.

Widyaningrum R ，Wu YW ，Delila L ，Lee DY ，Wang TJ ，Burnouf T ... -  《-》

Neuroprotective activity of a virus-safe nanofiltered human platelet lysate depleted of extracellular vesicles in Parkinson's disease and traumatic brain injury models.

Brain administration of human platelet lysates (HPL) is a potential emerging biotherapy of neurodegenerative and traumatic diseases of the central nervous system. HPLs being prepared from pooled platelet concentrates, thereby increasing viral risks, manufacturing processes should incorporate robust virus-reduction treatments. We evaluated a 19 ± 2-nm virus removal nanofiltration process using hydrophilic regenerated cellulose hollow fibers on the properties of a neuroprotective heat-treated HPL (HPPL). Spiking experiments demonstrated >5.30 log removal of 20-22-nm non-enveloped minute virus of mice-mock particles using an immuno-quantitative polymerase chain reaction assay. The nanofiltered HPPL (NHPPL) contained a range of neurotrophic factors like HPPL. There was >2 log removal of extracellular vesicles (EVs), associated with decreased expression of pro-thrombogenic phosphatidylserine and procoagulant activity. LC-MS/MS proteomics showed that ca. 80% of HPPL proteins, including neurotrophins, cytokines, and antioxidants, were still found in NHPPL, whereas proteins associated with some infections and cancer-associated pathways, pro-coagulation and EVs, were removed. NHPPL maintained intact neuroprotective activity in Lund human mesencephalic dopaminergic neuron model of Parkinson's disease (PD), stimulated the differentiation of SH-SY5Y neuronal cells and showed preserved anti-inflammatory function upon intranasal administration in a mouse model of traumatic brain injury (TBI). Therefore, nanofiltration of HPL is feasible, lowers the viral, prothrombotic and procoagulant risks, and preserves the neuroprotective and anti-inflammatory properties in neuronal pre-clinical models of PD and TBI.

Delila L ，Nebie O ，Le NTN ，Barro L ，Chou ML ，Wu YW ，Watanabe N ，Takahara M ，Buée L ，Blum D ，Devos D ，Burnouf T ... -  《Bioengineering & Translational Medicine》