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

BACKGROUND: 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. METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.

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.

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.

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.

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.

MicroRNA-155 inhibits dengue virus replication by inducing heme oxygenase-1-mediated antiviral interferon responses.

MicroRNAs (miRNAs) have been reported to directly alter the virus life cycle and virus-host interactions, and so are considered promising molecules for controlling virus infection. In the present study, we observed that miR-155 time-dependently downregulated upon dengue virus (DENV) infection. In contrast, exogenous overexpression of miR-155 appeared to limit viral replication in vitro, suggesting that the low levels of miR-155 would be beneficial for DENV replication. In vivo, overexpression of miR-155 protected ICR suckling mice from the life-threatening effects of DENV infection and reduced virus propagation. Further investigation revealed that the anti-DENV activity of miR-155 was due to target Bach1, resulting in the induction of the heme oxygenase-1 (HO-1)-mediated inhibition of DENV NS2B/NS3 protease activity, ultimately leading to induction of antiviral interferon responses, including interferon-induced protein kinase R (PKR), 2'-5'-oligoadenylate synthetase 1 (OAS1), OAS2, and OAS3 expression, against DENV replication. Collectively, our results provide a promising new strategy to manage DENV infection by modulation of miR-155 expression.

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.

Heat-treated human platelet pellet lysate modulates microglia activation, favors wound healing and promotes neuronal differentiation in vitro.

The neurorestorative efficacy of human platelet lysates in neurodegenerative disorders is still under investigation. Platelets prepared from standard and pathogen reduced platelet concentrates were pelletized, washed, concentrated, and subjected to freeze-thawing. The lysate was heated to 56°C for 30 min and characterized. Toxicity was evaluated using SH-SY5Y neuroblastoma, BV-2 microglial, and EA-hy926 endothelial cells. Inflammatory activity was tested by examining tumor necrosis factor (TNF) and cyclooxygenase (COX)-2 expressions by BV-2 microglia with or without stimulation by lipopolysaccharides (LPS). The capacity to stimulate wound healing was evaluated by a scratch assay, and the capacity to differentiate SH-SY5Y into neurons was also examined. Platelet lysates contained a range of neurotrophins. They were not toxic to SH-SY5Y, EA-hy926, or BV-2 cells, did not induce the expression of TNF or COX-2 inflammatory markers by BV-2 microglia, and decreased inflammation after LPS stimulation. They stimulated the wound closure in the scratch assay and induced SH-SY5Y differentiation as revealed by the increased length of neurites as well as ß3-tubulin and neurofilament staining. These data confirm the therapeutic potential of platelet lysates in the treatment of disorders of the central nervous system and support further evaluation as novel neurorestorative biotherapy in preclinical models.

Human platelet lysates for human cell propagation.

A pathogen-free and standardized xeno-free supplement of growth media is required for the ex vivo propagation of human cells used as advanced therapeutic medicinal products and for clinical translation in regenerative medicine and cell therapies. Human platelet lysate (HPL) made from therapeutic-grade platelet concentrate (PC) is increasingly regarded as being an efficient xeno-free alternative growth medium supplement to fetal bovine serum (FBS) for clinical-grade isolation and/or propagation of human cells. Most experimental studies establishing the superiority of HPL over FBS were conducted using mesenchymal stromal cells (MSCs) from bone marrow or adipose tissues. Data almost unanimously concur that MSCs expanded in a media supplemented with HPL have improved proliferation, shorter doubling times, and preserved clonogenicity, immunophenotype, in vitro trilineage differentiation capacity, and T-cell immunosuppressive activity. HPL can also be substituted for FBS when propagating MSCs from various other tissue sources, including Wharton jelly, the umbilical cord, amniotic fluid, dental pulp, periodontal ligaments, and apical papillae. Interestingly, HPL xeno-free supplementation is also proving successful for expanding human-differentiated cells, including chondrocytes, corneal endothelium and corneal epithelium cells, and tenocytes, for transplantation and tissue-engineering applications. In addition, the most recent developments suggest the possibility of successfully expanding immune cells such as macrophages, dendritic cells, and chimeric antigen receptor-T cells in HPL, further broadening its use as a growth medium supplement. Therefore, strong scientific rationale supports the use of HPL as a universal growth medium supplement for isolating and propagating therapeutic human cells for transplantation and tissue engineering. Efforts are underway to ensure optimal standardization and pathogen safety of HPL to secure its reliability for clinical-grade cell-therapy and regenerative medicine products and tissue engineering.

Nanofiltration of growth media supplemented with human platelet lysates for pathogen-safe xeno-free expansion of mesenchymal stromal cells.

BACKGROUND AIMS: Human platelet lysate can replace fetal bovine serum (FBS) for xeno-free ex vivo expansion of mesenchymal stromal cells (MSCs), but pooling of platelet concentrates (PCs) increases risks of pathogen transmission. We evaluated the feasibility of performing nanofiltration of platelet lysates and determined the impact on expansion of bone marrow-derived MSCs. METHODS: Platelet lysates were prepared by freeze-thawing of pathogen-reduced (Intercept) PCs suspended in 65% storage solution (SPP+) and 35% plasma, and by serum-conversion of PCs suspended in 100% plasma. Lysates were added to the MSC growth media at 10% (v/v), filtered and subjected to cascade nanofiltration on 35- and 19-nm Planova filters. Media supplemented with 10% starting platelet lysates or FBS were used as the controls. Impacts of nanofiltration on the growth media composition, removal of platelet extracellular vesicles (PEVs) and MSC expansion were evaluated. RESULTS: Nanofiltration did not detrimentally affect contents of total protein and growth factors or the biochemical composition. The clearance factor of PEVs was >3 log values. Expansion, proliferation, membrane markers, differentiation potential and immunosuppressive properties of cells in nanofiltered media were consistently better than those expanded in FBS-supplemented media. Compared with FBS, chondrogenesis and osteogenesis genes were expressed more in nanofiltered media, and there were fewer senescent cells over six passages. CONCLUSIONS: Nanofiltration of growth media supplemented with two types of platelet lysates, including one prepared from pathogen-reduced PCs, is technically feasible. These data support the possibility of developing pathogen-reduced xeno-free growth media for clinical-grade propagation of human cells.

Clinical-grade cryopreserved doxorubicin-loaded platelets: role of cancer cells and platelet extracellular vesicles activation loop.

BACKGROUND: Human platelets (PLT) and PLT-extracellular vesicles (PEV) released upon thrombin activation express receptors that interact with tumour cells and, thus, can serve as a delivery platform of anti-cancer agents. Drug-loaded nanoparticles coated with PLT membranes were demonstrated to have improved targeting efficiency to tumours, but remain impractical for clinical translation. PLT and PEV targeted drug delivery vehicles should facilitate clinical developments if clinical-grade procedures can be developed. METHODS: PLT from therapeutic-grade PLT concentrate (PC; N > 50) were loaded with doxorubicin (DOX) and stored at - 80 °C (DOX-loaded PLT) with 6% dimethyl sulfoxide (cryopreserved DOX-loaded PLT). Surface markers and function of cryopreserved DOX-loaded PLT was confirmed by Western blot and thromboelastography, respectively. The morphology of fresh and cryopreserved naïve and DOX-loaded PLT was observed by scanning electron microscopy. The content of tissue factor-expressing cancer-derived extracellular vesicles (TF-EV) present in conditioned medium (CM) of breast cancer cells cultures was measured. The drug release by fresh and cryopreserved DOX-loaded PLT triggered by various pH and CM was determined by high performance liquid chromatography. The thrombin activated PEV was analyzed by nanoparticle tracking analysis. The cellular uptake of DOX from PLT was observed by deconvolution microscopy. The cytotoxicities of DOX-loaded PLT, cryopreserved DOX-loaded PLT, DOX and liposomal DOX on breast, lung and colon cancer cells were analyzed by CCK-8 assay. RESULTS: 15~36 × 106 molecules of DOX could be loaded in each PLT within 3 to 9 days after collection. The characterization and bioreactivity of cryopreserved DOX-loaded PLT were preserved, as evidenced by (a) microscopic observations, (b) preservation of important PLT membrane markers CD41, CD61, protease activated receptor-1, (c) functional activity, (d) reactivity to TF-EV, and (e) efficient generation of PEV upon thrombin activation. The transfer of DOX from cryopreserved PLT to cancer cells was achieved within 90 min, and stimulated by TF-EV and low pH. The cryopreserved DOX-loaded PLT formulation was 7~23-times more toxic to three cancer cells than liposomal DOX. CONCLUSIONS: Cryopreserved DOX-loaded PLT can be prepared under clinically compliant conditions preserving the membrane functionality for anti-cancer therapy. These findings open perspectives for translational applications of PLT-based drug delivery systems.

A double-virally-inactivated (Intercept-solvent/detergent) human platelet lysate for in vitro expansion of human mesenchymal stromal cells.

BACKGROUND: Pooled human platelet lysate (HPL) can replace fetal bovine serum (FBS) as xeno-free supplement for ex vivo expansion of mesenchymal stromal cells (MSCs). We evaluate here whether a double-virally-inactivated HPL (DVI-HPL) prepared from expired Intercept-treated platelet concentrates (PCs) and treated by solvent/detergent (S/D) can be used for MSC expansion. STUDY DESIGN AND METHODS: Expired Intercept-treated PCs in 65% platelet (PLT) additive solution were pooled and subjected to a 1% tri-n-butyl phosphate/1% Triton X-45 treatment followed by soybean oil, hydrophobic interaction chromatography purification, and sterile filtration. Bone marrow-derived MSCs (BM-MSCs) were expanded for four passages in growth medium containing 10% DVI-HPL, I-HPL (from Intercept-PC only), untreated HPL, and FBS. MSC morphology, doubling time, immunophenotype, immunosuppressive activity, and differentiation capacity were compared. RESULTS: Expanded cells had typical spindle morphology and showed higher viability in all HPL conditions than in FBS. The DVI-HPL and FBS-expanded cells were morphologically larger than in I-HPL and HPL supplements. The cumulative population doubling was lower using DVI-HPL than with HPL and I-HPL, but significantly higher than using FBS. Immunophenotype was not affected by the supplements used. Immunosuppressive activity was maintained with all supplements. Differentiation capacity into chondrocytes and osteocytes was more effective in DVI-HPL but less toward adipocytes compared to other supplements. CONCLUSIONS: Human PLT lysate made from Intercept-PCs subjected to S/D treatment may be an alternative to untreated HPL and to I-HPL for BM-MSC expansion. This finding reinforces the potential of HPL as a virally safe alternative to FBS for clinical grade MSC expansion protocols.

Viral safety of human platelet lysate for cell therapy and regenerative medicine: Moving forward, yes, but without forgetting the past.

Growth factor-rich pooled human platelet lysate (HPL), made from human platelet concentrates, is one new blood-derived bioproduct that is attracting justified interest as a xeno-free supplement of growth media for human cell propagation for cell therapy. HPL can also find potentially relevant applications in the field of regenerative medicine. Therefore, the therapeutic applications of HPL go far beyond the standard clinical applications of the traditional blood products typically used in patients suffering from life-threatening congenital or acquired deficiencies in cellular components or proteins due to severe genetic diseases or trauma. A wider population of patients, suffering from various pathologies than has traditionally been the case, is thus, now susceptible to receiving a human blood-derived product. These patients would, therefore, be exposed to the possible, but avoidable, side effects of blood products, including transfusion-transmitted infections, most specifically virus transmissions. Unfortunately, not all manufacturers, suppliers, and users of HPL may have a strong background in the blood product industry. As such, they may not be fully aware of the various building blocks that should contribute to the viral safety of HPL as is already the case for any licensed blood products. The purpose of this manuscript is to reemphasize all the measures, including in regulatory aspects, capable of assuring that HPL exhibits a sufficient pathogen safety margin, especially when made from large pools of human platelet concentrates. It is vital to remember the past to avoid that the mistakes, which happened 30 to 40 years ago and led to the contamination of many blood recipients, be repeated due to negligence or ignorance of the facts.

Adhesion of a polymer-grafted nanoparticle to cells explored using generalized Langevin dynamics.

We model a polymer-grafted stealth nanoparticle (SNP) as a composite system consisting of a spherical core coated with a porous polymeric brush with end-ligands. Adjacent to target cells, the near-wall hydrodynamics, thermal fluctuations, and thermodynamic adhesive interactions simultaneously impact the transient motion of the SNP. Employing both the Langevin framework for the effective hard sphere dynamics and the coupled generalized Langevin framework for the nanoparticle-polymer dynamics, we comprehensively investigate the velocity and position temporal relaxations of the SNP in the absence and presence of end-to-end distance fluctuations for the tethered polymer. We demonstrate that polymer structural relaxations substantially impact the SNP adhesive dynamics, especially when the grafted polymer is more flexible. Moreover, a long-time tail with t-3/2 scaling due to polymer chain-length fluctuations is observed in the velocity autocorrelation for a bound SNP. Finally, the thermodynamic effects of membrane morphology on SNP adhesion are explored by modifying the membrane-mediated binding potential of mean force.

Triglyceride to high-density lipoprotein cholesterol ratio and carotid intima-medial thickness in Chinese adolescents with newly diagnosed type 2 diabetes mellitus.

OBJECTIVE: To investigate the relationship between triglyceride to high-density lipoprotein cholesterol (TG/HDL-C) ratio and carotid intima-medial thickness (CIMT) in Chinese youth and adolescents with newly diagnosed type 2 diabetes mellitus (T2DM). METHODS: Ninety-eight subjects aged 10-24 yr with newly-diagnosed T2DM had general inflammation, anthropometric, laboratory and CIMT data collected, and were divided into three groups based on TG/HDL-C tertiles. RESULTS: There were no significant differences in gender, age, fasting plasma glucose (FPG), hemoglobin A1c (HbA1c), and carotid arterial diameter (CAD) among the groups based on TG/HDL-C tertiles. Across TG/HDL-C tertiles, there was a significant progressive increase in body mass index (BMI), systolic blood pressure (SBP), diastolic blood pressure (DBP), homeostasis model assessment-estimated insulin resistance (HOMA-IR), TG, total cholesterol (TC), low-density lipoprotein-cholesterol (LDL-C) and CIMT (all P < 0.01 or P < 0.05), while HDL-C was decreased significantly across the groups (P < 0.01). In general linear regression model, TG/HDL-C was an independent determinant of CIMT even after adjusting for BMI, SBP, DBP, TG, TC, LDL-C, HDL-C, HbA1c and HOMA-IR. CONCLUSION: TG/HDL-C ratio, the marker of small dense LDL particles, is an independent determinant of CIMT in Chinese youth and adolescents with newly diagnosed T2DM, and may be a simple and helpful tool in predicting the increased CIMT in such patients.

Smart blood cell and microvesicle-based Trojan horse drug delivery: Merging expertise in blood transfusion and biomedical engineering in the field of nanomedicine.

Therapeutic and diagnostic applications of nanomedicine are playing increasingly important roles in human health. Various types of synthetic nanoparticles, including liposomes, micelles, and other nanotherapeutic platforms and conjugates, are being engineered to encapsulate or carry drugs for treating diseases such as cancer, cardiovascular disorders, neurodegeneration, and inflammations. Nanocarriers are designed to increase the half-life of drugs, decrease their toxicity and, ideally, target pathological sites. Developing smart carriers with the capacity to deliver drugs specifically to the microenvironment of diseased cells with minimum systemic toxicity is the goal. Blood cells, and potentially also the liposome-like micro- and nano-vesicles they generate, may be regarded as ideally suited to perform such specific targeting with minimum immunogenic risks. Blood cell membranes are "decorated" with complex physiological receptors capable of targeting and communicating with other cells and tissues and delivering their content to the surrounding pathological microenvironment. Blood cells, such as erythrocytes, have been developed as permeable carriers to release drugs to diseased tissues or act as biofactory allowing enzymatic degradation of a pathological substrate. Interestingly, attempts are also being made to improve the targeting capacity of synthetic nanoparticles by "decorating" their surface with blood cell membrane receptor-like biochemical structures. Research is needed to further explore the benefits that blood cell-derived microvesicles, as a Trojan horse delivery systems, can bring to the arsenal of therapeutic micro- and nanotechnologies. This short review focuses on the therapeutic roles that red blood cells and platelets can play as smart drug-delivery systems, and highlights the benefits that blood transfusion expertise can bring to this exciting and novel biomedical engineering field.

Dedicated removal of immunoglobulin (Ig)A, IgM, and Factor (F)XI/activated FXI from human plasma IgG.

BACKGROUND: Adverse events can be associated with treating critically ill patients with immunoglobulin (Ig)G. Some adverse events are due to contaminants like IgA and activated Factor (F)XI. Therefore, new purification strategies are needed for dedicated removal of these contaminants without impairing IgG recovery. STUDY DESIGN AND METHODS: An immunoglobulin fraction containing IgG, IgM, and IgA was prepared by caprylic acid precipitation of cryoprecipitate-poor plasma. The capacities of the cation exchangers (S HyperCel and CM Ceramic HyperD F) and anion exchangers (HyperCel STAR AX and Q HyperCel) to remove IgA, IgM, and spiked FXI were tested following a design of experiment approach using microplates and chromatographic column scale-up. FXI removal was also evaluated using Mustang S chromatographic membranes. IgG/IgG subclasses, IgA, IgM, and FXI were assessed by enzyme-linked immunosorbent assay, and caprylic acid, by gas chromatography. RESULTS: Extensive removal of IgA and IgM, but not FXI, was achieved by a two-step chromatographic process combining S HyperCel used in the IgG binding and elution mode and HyperCel STAR AX used in the IgG flow-through mode, providing high IgG and IgG subclass recovery (>85%), high purity (>99.5%), and efficient removal of IgA (<0.5%) and IgM (undetectable). Twenty-six-fold FXI removal was achieved by processing the resulting purified IgG fraction through Mustang S cation-exchanger membranes at pH 6.0 and 12.7 mS/cm. Caprylic acid was removed by S HyperCel. CONCLUSIONS: Combining S HyperCel and HyperCel STAR AX extensively removed IgA and IgM, with good IgG recovery. Mustang® S membranes can be used for dedicated removal of FXI.

CTRP3 modulates the expression and secretion of adipokines in 3T3-L1 adipocytes.

The objective of this study was to investigate the impact of C1q/TNF related protein 3 (CTRP3), a novel adipokine, on the expression and secretion of adiponectin, leptin, visfatin, and apelin in 3T3-L1 adipocytes. The effect of insulin resistance on the impact was also investigated. 3T3-L1 adipocytes were treated with different concentrations (0, 10, 50, 250, 1250 ng/mL) CTRP3 for 12 h, and with 250 ng/mL CTRP3 for different times (0, 6, 12, 24, 48 h). The expression of adipokines between normal and insulin resistant adipocytes, as well as between the adipocytes pre-treated with and without Compound C were compared. The secretion and gene expression of the adipokines were detected by enzyme-linked immunosorbent assay (ELISA) and real-time polymerase chain reaction (RT-PCR), respectively. The relative expression of AMPK (thr172) was detected by western blot analysis. With the increase in CTRP3 concentration or the duration of the treatment, the secretion of adiponectin, leptin, visfatin and apelin were all increased accordingly, which was significant under the treatment with 250 ng/mL and 1250 ng/mL CTRP3 for 12 h as well as 250 ng/mL CTRP3 for 12 h, 24 h and 48 h. Gene expression showed a similar trend. The secretion and gene expression of adipokines in insulin resistant adipocytes were all decreased significantly in comparison with that of normal adipocytes. The secretion secretion and gene expression of adiponectin, and the relative expression of AMPK (thr172) in adipocytes pre-treated with Compound C were decreased significantly in comparison with that in adipocytes without Compound C pretreatment. Thus, CTRP3 increased the expression and secretion of adiponectin, leptin, visfatin, and apelin in 3T3-L1 adipocytes, while insulin resistance inhibited the effects. CTRP3 up-regulated the expression of adiponectin in 3T3-L1 adipocytes through AMPK signaling pathway.

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.

Cognitive protection and brain entropy changes from omega-3 polyunsaturated fatty acids supplement in late-life depression: A 52-week randomized controlled trial.

BACKGROUND: Late-life depression (LLD) is associated with risk of dementia, yet intervention of LLD provides an opportunity to attenuate subsequent cognitive decline. Omega-3 polyunsaturated fatty acids (PUFAs) supplement is a potential intervention due to their beneficial effect in depressive symptoms and cognitive function. To explore the underlying neural mechanism, we used resting-state functional MRI (rs-fMRI) before and after omega-3 PUFAs supplement in older adults with LLD. METHODS: A 52-week double-blind randomized controlled trial was conducted. We used multi-scale sample entropy to analyze rs-fMRI data. Comprehensive cognitive tests and inflammatory markers were collected to correlate with brain entropy changes. RESULTS: A total of 20 patients completed the trial with 11 under omega-3 PUFAs and nine under placebo. While no significant global cognitive improvement was observed, a marginal enhancement in processing speed was noted in the omega-3 PUFAs group. Importantly, participants receiving omega-3 PUFAs exhibited decreased brain entropy in left posterior cingulate gyrus (PCG), multiple visual areas, the orbital part of the right middle frontal gyrus, and the left Rolandic operculum. The brain entropy changes of the PCG in the omega-3 PUFAs group correlated with improvement of language function and attenuation of interleukin-6 levels. LIMITATIONS: Sample size is small with only marginal clinical effect. CONCLUSION: These findings suggest that omega-3 PUFAs supplement may mitigate cognitive decline in LLD through anti-inflammatory mechanisms and modulation of brain entropy. Larger clinical trials are warranted to validate the potential therapeutic implications of omega-3 PUFAs for deterring cognitive decline in patients with late-life depression.