Proteomics of human platelet lysates and insight from animal studies on platelet protein diffusion to hippocampus upon intranasal administration.

Human platelet lysates (HPLs) from allogeneic platelet concentrates (PCs) are biomaterials, which are rich in various trophic factors, increasingly used in regenerative medicine and biotherapy. Understanding how preparation methods influence the HPL protein profile, biological function, and clinical outcomes is crucial. Our study sheds light on the proteomes and functionality of different HPLs, with the aim of advancing their scientifically grounded clinical applications. To achieve this, PCs suspended in plasma underwent three distinct processing methods, resulting in seven HPL types. We used three characterization techniques: label-free proteomics and tandem mass tag (TMT)-based quantitative proteomics, both before and after the immunodepletion of abundant plasma proteins. Bioinformatic tools assessed the proteome, and western blotting validated our quantitative proteomics data. Subsequent pre-clinical studies with fluorescent labeling and label-free proteomics were used as a proof of concept for brain diffusion. Our findings revealed 1441 proteins detected using the label-free method, 952 proteins from the TMT experiment before and after depletion, and 1114 proteins from the subsequent TMT experiment on depleted HPLs. Most detected proteins were cytoplasmic, playing key roles in catalysis, hemostasis, and immune responses. Notably, the processing methodologies significantly influenced HPL compositions, their canonical pathways, and, consequently, their functionality. Each HPL exhibited specific abundant proteins, providing valuable insight for tailored clinical applications. Immunoblotting results for selected proteins corroborated our quantitative proteomics data. The diffusion and differential effects to the hippocampus of a neuroprotective HPL administered intranasally to mice were demonstrated. This proteomics study advances our understanding of HPLs, suggesting ways to standardize and customize their production for better clinical efficacy in regenerative medicine and biotherapy. Proteomic analyses also offered objective evidence that HPPL, upon intranasal delivery, not only effectively diffuses to the hippocampus but also alters protein expression in mice, bolstering its potential as a treatment for memory impairments.

Platelet extracellular vesicles are efficient delivery vehicles of doxorubicin, an anti-cancer drug: preparation and in vitro characterization.

Platelet extracellular vesicles (PEVs) are an emerging delivery vehi for anticancer drugs due to their ability to target and remain in the tumor microenvironment. However, there is still a lack of understanding regarding yields, safety, drug loading efficiencies, and efficacy of PEVs. In this study, various methods were compared to generate PEVs from clinical-grade platelets, and their properties were examined as vehicles for doxorubicin (DOX). Sonication and extrusion produced the most PEVs, with means of 496 and 493 PEVs per platelet (PLT), respectively, compared to 145 and 33 by freeze/thaw and incubation, respectively. The PEVs were loaded with DOX through incubation and purified by chromatography. The size and concentration of the PEVs and PEV-DOX were analyzed using dynamic light scattering and nanoparticle tracking analysis. The results showed that the population sizes and concentrations of PEVs and PEV-DOX were in the ranges of 120-150 nm and 1.2-6.2 × 1011 particles/mL for all preparations. The loading of DOX determined using fluorospectrometry was found to be 2.1 × 106, 1.7 × 106, and 0.9 × 106 molecules/EV using freeze/thaw, extrusion, and sonication, respectively. The internalization of PEVs was determined to occur through clathrin-mediated endocytosis. PEV-DOX were more efficiently taken up by MDA-MB-231 breast cancer cells compared to MCF7/ADR breast cancer cells and NIH/3T3 cells. DOX-PEVs showed higher anticancer activity against MDA-MB-231 cells than against MCF7/ADR or NIH/3T3 cells and better than acommercial liposomal DOX formulation. In conclusion, this study demonstrates that PEVs generated by PLTs using extrusion, freeze/thaw, or sonication can efficiently load DOX and kill breast cancer cells, providing a promising strategy for further evaluation in preclinical animal models. The study findings suggest that sonication and extrusion are the most efficient methods to generate PEVs and that PEVs loaded with DOX exhibit significant anticancer activity against MDA-MB-231 breast cancer cells.

What is the context?● Current synthetic drug delivery systems can have limitations and side effects.● Platelet extracellular vesicles (PEVs) are a natural and potentially safer alternative for delivering cancer drugs to tumors.● However, there is still a lack of understanding about how to produce PEVs and how effective they are in delivering drugs.What is new?● We compared different methods for producing PEVs from clinical-grade platelets and found that sonication and extrusion were the most effective methods.● The PEVs were loaded with a cancer drug called doxorubicin (DOX) and tested their ability to kill breast cancer cells.What is the impact?● PEVs loaded with DOX were effective at killing cancer cells, especially MDA-MB-231 breast cancer cells.● This study demonstrates that PEVs are a promising strategy for delivering cancer drugs to tumors and that sonication and extrusion are the most efficient methods for producing PEVs.● The results suggest that further evaluation of PEVs in preclinical animal models is warranted to determine their potential as a cancer drug delivery system.Abbreviations: ADP: adenosine diphosphate; bFGF: basic fibroblast growth factor; BSA: bovine serum albumin; CD41: platelet glycoprotein IIb; CD62P: P-selectin; CFDASE: 5-(and-6)-carboxyfluorescein diacetate: succinimidyl ester; CPLT: cryopreserved platelet; CPZ: chlorpromazine hydrochloride; CTC: circulating tumor cell; DMSO: dimethyl sulfoxide; DDS: drug delivery system; DOX: doxorubicin; EPR: enhanced permeability and retention; EV: extracellular vesicle; FBS: fetal bovine serum; GMP: good manufacturing practice; GF: growth factor; HER2: human epidermal growth factor receptor 2; HGF: hepatocyte growth factor; Lipo-DOX: liposomal doxorubicin; MDR: multi-drug resistance; MMP-2: matrix metalloproteinase-2; MP: microparticle; MSC: mesenchymal stromal cell; NP: nanoparticle; NTA: nanoparticle tracking analysis; PAR-1: protease activated receptor-1; PAS: platelet additive solution; PBS: phosphate-buffered saline; PC: platelet concentrate; PEG: polyethylene glycol; PEV: platelet-derived extracellular vesicle; DOX-PEV: doxorubicin-loaded platelet-derived extracellular vesicle-encapsulated; PFA: paraformaldehyde; PF4: platelet factor 4; P-gp: P-glycoprotein; PLT: platelet; PS: phosphatidylserine; SDS-PAGE: sodium dodecylsulfate polyacrylamide gel electrophoresis; SEM: scanning electron microscopy; TCIPA: tumor cell-induced PLT aggregation; TDDS: targeted drug delivery system; TEG: thromboelastography; TF: tissue factor; TF-EV: extracellular vesicle expressing tissue factor; TME: tumor microenvironment; TNBC: triple-negative breast cancer; TXA2: thromboxane-A2; VEGF: vascular endothelial growth factor; WHO: World Health Organization.

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.

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

Corneal endothelial cells (CECs) slowly decrease in number with increasing age, which is a clinical issue as these cells have very limited regenerative ability. Therapeutic platelet biomaterials are increasingly used in regenerative medicine and cell therapy because of their safety, cost-effective manufacture, and global availability from collected platelet concentrates (PCs). Platelet extracellular vesicles (PEVs) are a complex mixture of potent bioactive vesicles rich in molecules believed to be instrumental in tissue repair and regeneration. In this study we investigated the feasibility of using a PEVs preparation as an innovative regenerative biotherapy for corneal endothelial dysfunction. The PEVs were isolated from clinical-grade human PC supernatants by 20,000 × g ultracentrifugation and resuspension. PEVs exhibited a regular, fairly rounded shape, with an average size of <200 nm and were present at a concentration of approximately 1011 /mL. PEVs expressed cluster of differentiation 41 (CD41) and CD61, characteristic platelets membrane markers, and CD9 and CD63. ELISA and LC-MS/MS proteomic analyses revealed that the PEVs contained mixtures of growth factors and multiple other trophic factors, as well as proteins related to extracellular exosomes with functional activities associated with cell cadherin and adherens pathways. CECs treated with PEVs showed increased viability, an enhanced wound-healing rate, stronger proliferation markers, and an improved adhesion rate. PEVs did not exert cellular toxicity as evidenced by the maintenance of cellular morphology and preservation of corneal endothelial proteins. These findings clearly support further investigations of PEV biomaterials in animal models for translation as a new CEC regeneration biotherapy.

Characterization and Chromatographic Isolation of Platelet Extracellular Vesicles from Human Platelet Lysates for Applications in Neuroregenerative Medicine.

Human platelet lysates (HPLs) made from clinical-grade platelet concentrates are currently evaluated in the preclinical models of Parkinson's disease, Alzheimer's disease, traumatic brain injury, and others, as a new polyvalent neuroprotective biotherapy of the central nervous system. However, the presence and content of extracellular vesicles (EVs) in HPLs and their potential contribution to the neuroprotective and neurorestorative activities of HPLs are still unknown. We, therefore, characterized the EVs present in four different HPL preparations and after purification by size-exclusion chromatography. We then tested the effect of the isolated EVs on neuronal cell repair. We identified that all four HPLs contained a high and similar amount of EVs (1011 to 1012/mL) with a mean size ranging from ca. 50 to 300 nm and a negative zeta potential as determined by nanoparticle tracking analysis and dynamic light scattering. Western blot analysis revealed that the EVs present in HPLs expressed the clusters of differentiation 41 (CD41) and 61 (CD61) characteristic of platelets. These EVs were efficiently isolated from HPL proteins by Sepharose CL-2B size-exclusion column chromatography as confirmed by total protein determination and protein profile by sodium dodecyl sulfate polyacrylamide gel electrophoresis, with 73-85% recovery and maintenance of their size, negative zeta potential, and CD41 and CD61 expression. Interestingly, the EVs purified from the four HPLs exhibited a differential capacity to promote cell growth and migration in a wound-healing assay using SH-SY5Y neuronal cells, and one EV preparation stimulated network formation in primary neuronal cultures. These data indicated that the EVs present in HPLs have different neuroregenerative capacities and that some EV preparations may have interesting applications as a stand-alone therapy for usage in neuroregenerative medicine.

A purified human platelet pellet lysate rich in neurotrophic factors and antioxidants repairs and protects corneal endothelial cells from oxidative stress.

Human platelet lysate (HPL) is a complex mixture of potent bioactive molecules instrumental in tissue repair and regeneration. Due to their remarkable safety, cost-effective production, and availability at global level from collected platelet concentrates, HPLs can become a powerful biotherapy for various therapeutic applications, if standardized and carefully validated through pre-clinical and clinical studies. In this work, the possibility to use a tailor-made HPL as a corneal transplant alternative to treat the gradual decrease in the number of corneal endothelial cells (CECs) associated with aging, was evaluated. The HPL preparation was thoroughly characterized using various proteomics tools that revealed a remarkable richness in multiple growth factors and antioxidants. Treatment of B4G12 and BCE C/D-1b CECs with the HPL increased their viability, enhanced the wound closure rate, and maintained cell growth and typical hexagonal morphology. Besides, this HPL significantly protected against tert-butyl hydroperoxide (TBHP)-induced oxidative stress as evidenced by increasing CEC viability, decreased cell death and reactive oxygen species formation, and enhanced antioxidant capacity. Proteomics analysis of treated CECs confirmed that HPL treatment triggered the corneal healing pathway and enhanced oxidative stress. These data strongly support further pre-clinical evaluation of this tailor-made HPL as a novel CEC regeneration biotherapy. HPL treatment may eventually represent a pragmatic and cost-effective alternative to corneal transplant to treat damages of the corneal endothelium which is a major cause of blindness worldwide.

Removal of minute virus of mice-mock virus particles by nanofiltration of culture growth medium supplemented with 10% human platelet lysate.

BACKGROUND AIMS: Platelet concentrates (PCs) are pooled to prepare human platelet lysate (HPL) supplements of growth media to expand primary human cells for transplantation; this increases the risk of contamination by known, emerging, and unknown viruses. This possibility should be of concern because viral contamination of cell cultures is difficult to detect and may have detrimental consequences for recipients of cell therapies. Viral reduction treatments of chemically defined growth media have been proposed, but they are not applicable when media contain protein supplements currently needed to expand primary cell cultures. Recently, we successfully developed a Planova 35NPlanova 20N nanofiltration sequence of growth media supplemented with two types of HPL. The nanofiltered medium was found to be suitable for mesenchymal Stromal cell (MSC) expansion. METHODS: Herein, we report viral clearance achieved by this nanofiltration process used for assessing a new experimental model using non-infectious minute virus of mice-mock virus particle (MVM-MVP) and its quantification by an immunoqPCR. Then, high doses of MVM-MVP (1012 MVPs/mL) were spiked to obtain a final concentration of 1010 MVPs/mL in Planova 35N-nanofiltered growth medium supplemented with both types of HPLs [serum converted platelet lysate SCPL) and intercept human platelet lysate (I-HPL)] at 10% (v/v) and then filtering through Planova 20N. RESULTS: No substantial interference of growth medium matrices by the immune-qPCR assay was first verified. Log reduction values (LRVs) were ≥ 5.43 and ≥ 5.36 respectively, SCPL and I-HPL media. MVM-MVPs were also undetectable by dynamic light scattering and transmission electron microscopy. CONCLUSIONS: The nanofiltration of growth media supplemented with 10% HPL provides robust removal of small nonenveloped viruses, and is an option to improve the safety of therapeutic cells expanded using HPL supplements.

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.

Extensive characterization of the composition and functional activities of five preparations of human platelet lysates for dedicated clinical uses.

Human platelet lysates (HPLs), rich in various growth factors and cell growth-promoting molecules, encompass a new range of blood products that are being used for regenerative medicine, cell therapies, and tissue engineering. Well-characterized dedicated preparations, tailor-made to best fit specific therapeutic applications, are needed for optimal clinical efficacy and safety. Here, five types of HPL were prepared from the same platelet concentrates and extensively characterized to determine and compare their proteins, growth factors, cytokines, biochemical profiles, thrombin-generating capacities, thrombin-associated proteolytic activities, phospholipid-associated procoagulant potential, contents of extracellular vesicles expressing phosphatidylserine and tissue factor, and antioxidative properties. Our results revealed that all five HPL preparations contained detectable supraphysiological levels, in the ca. 0.1 ~ 350-ng/ml range, of all growth factors assessed, except insulin-like growth factor-1 detected only in HPL containing plasma. There were significant differences observed among these HPLs in total protein content, fibrinogen, complement components C3 and C4, albumin, and immunoglobulin G, and, most importantly, in their functional coagulant and procoagulant activities and antioxidative capacities. Our data revealed that the biochemical and functional properties of HPL preparations greatly vary depending upon their mode of production, with potential impacts on the safety and efficacy for certain clinical indications. Modes of preparation of HPLs should be carefully designed, and the product properties carefully evaluated based on the intended therapeutic use to ensure optimal clinical outcomes.