Methods,Mechanisms,and Application Prospects for Enhancing Extracellular Vesicle Uptake

Extracellular vesicles(EVs)are considered to be a new generation of bioinspired nanoscale drug delivery systems due to their low immunogenicity,natural functionality,and excellent biocompatibility.However,limitations such as low uptake efficiency,insufficient production,and inhomogeneous performance undermine their potential.To address these issues,numerous researchers have put forward various methods and applications for enhancing EV uptake in recent decades.In this review,we introduce various methods for the cellular uptake of EVs and summarize recent advances on the methods and mechanisms for enhancing EV uptake.In addition,we provide further understanding regarding enhancing EV uptake and put forward prospects and challenges for the development of EV-based therapy in the future.

Pathological and therapeutic effects of extracellular vesicles in neurological and neurodegenerative diseases

Extracellular vesicles are released by all cell types and contain proteins,microRNAs,mRNAs,and other bioactive molecules.Extracellular vesicles play an important role in intercellular communication and in the modulation of the immune system and neuroinflammation.The cargo of extra cellular vesicles(e.g.,proteins and microRNAs)is altered in pathological situations.Extracellular vesicles contribute to the pathogenesis of many pathologies associated with sustained inflammation and neuroinflammation,including cance r,diabetes,hype rammonemia and hepatic encephalopathy,and other neurological and neurodegenerative diseases.Extracellular vesicles may cross the blood-brain barrier and transfer pathological signals from the periphery to the brain.This contributes to inducing neuroinflammation and cognitive and motor impairment in hyperammonemia and hepatic encephalopathy and in neurodegenerative diseases.The mechanisms involved are beginning to be unde rstood.For example,increased tumor necrosis factor a in extracellular vesicles from plasma of hype rammonemic rats induces neuroinflammation and motor impairment when injected into normal rats.Identifying the mechanisms by which extracellular vesicles contribute to the pathogenesis of these diseases will help to develop new treatments and diagnostic tools for their easy and early detection.In contrast,extra cellular vesicles from mesenchymal stem cells have therapeutic utility in many of the above pathologies,by reducing inflammation and neuroinflammation and improving cognitive and motor function.These extra cellular vesicles recapitulate the beneficial effects of mesenchymal stem cells and have advantages as therapeutic tools:they are less immunoge nic,may not diffe rentiate to malignant cells,cross the blood-brain barrier,and may reach more easily target organs.Extracellular vesicles from mesenchymal stem cells have beneficial effects in models of ischemic brain injury,Alzheimer's and Parkinson's diseases,hyperammonemia,and hepatic encephalopathy.Extracellular vesicles from mesenchymal stem cells modulate the immune system,promoting the shift from a pro-inflammato ry to an anti-inflammatory state.For example,extracellular vesicles from mesenchymal stem cells modulate the Th17/Treg balance,promoting the anti-inflammatory Treg.Extracellular vesicles from mesenchymal stem cells may also act directly in the brain to modulate microglia activation,promoting a shift from a pro-inflammatory to an anti-inflammatory state.This reduces neuroinflammation and improves cognitive and motor function.Two main components of extracellular vesicles from mesenchymal stem cells which contribute to these beneficial effects are transforming growth factor-βand miR-124.Identifying the mechanisms by which extracellular vesicles from mesenchymal stem cells induce the beneficial effects and the main molecules(e.g.,proteins and mRNAs)involved may help to improve their therapeutic utility.The aims of this review are to summarize the knowledge of the pathological effects of extracellular vesicles in different pathologies,the therapeutic potential of extra cellular vesicles from mesenchymal stem cells to recover cognitive and motor function and the molecular mechanisms for these beneficial effects on neurological function.

Mesenchymal stem cell-derived extracellular vesicles as a cell-free therapy for traumatic brain injury via neuroprotection and neurorestoration

Traumatic brain injury is a serious and complex neurological condition that affects millions of people worldwide.Despite significant advancements in the field of medicine,effective treatments for traumatic brain injury remain limited.Recently,extracellular vesicles released from mesenchymal stem/stromal cells have emerged as a promising novel therapy for traumatic brain injury.Extracellular vesicles are small membrane-bound vesicles that are naturally released by cells,including those in the brain,and can be engineered to contain therapeutic cargo,such as anti-inflammatory molecules,growth factors,and microRNAs.When administered intravenously,extra cellular vesicles can cross the blood-brain barrier and deliver their cargos to the site of injury,where they can be taken up by recipient cells and modulate the inflammatory response,promote neuroregeneration,and improve functional outcomes.In preclinical studies,extracellular vesicle-based therapies have shown promising results in promoting recove ry after traumatic brain injury,including reducing neuronal damage,improving cognitive function,and enhancing motor recovery.While further research is needed to establish the safety and efficacy of extra cellular vesicle-based therapies in humans,extra cellular vesicles represent a promising novel approach for the treatment of traumatic brain injury.In this review,we summarize mesenchymal ste m/stromal cell-de rived extracellular vesicles as a cell-free therapy for traumatic brain injury via neuroprotection and neurorestoration and brainderived extracellular vesicles as potential biofluid biomarkers in small and large animal models of traumatic brain injury.

Neutrophil extracellular traps mediate neuro-immunothrombosis

Neutrophil extracellular traps are primarily composed of DNA and histones and are released by neutrophils to promote inflammation and thrombosis when stimulated by various inflammato ry reactions.Neutrophil extracellular trap formation occurs through lytic and non-lytic pathways that can be further classified by formation mechanisms.Histones,von Willebrand factor,fibrin,and many other factors participate in the interplay between inflammation and thrombosis.Neuroimmunothrombosis summarizes the intricate interplay between inflammation and thrombosis during neural development and the pathogenesis of neurological diseases,providing cutting-edge insights into post-neurotrauma thrombotic events.The blood-brain barrier defends the brain and spinal cord against external assaults,and neutrophil extracellular trap involvement in blood-brain barrier disruption and immunothrombosis contributes substantially to secondary injuries in neurological diseases.Further research is needed to understand how neutrophil extracellular traps promote blood-brain barrier disruption and immunothrombosis,but recent studies have demonstrated that neutrophil extracellular traps play a crucial role in immunothrombosis,and identified modulators of neuro-immunothrombosis.However,these neurological diseases occur in blood vessels,and the mechanisms are unclear by which neutrophil extracellular traps penetrate the blood-brain barrier to participate in immunothrombosis in traumatic brain injury.This review discusses the role of neutrophil extracellular traps in neuro-immunothrombosis and explores potential therapeutic interventions to modulate neutrophil extracellular traps that may reduce immunothrombosis and improve traumatic brain injury outcomes.

Connecting cellular mechanisms and extracellular vesicle cargo in traumatic brain injury

Traumatic brain injury is followed by a cascade of dynamic and complex events occurring at the cellular level. These events include: diffuse axonal injury, neuronal cell death, blood-brain barrier break down, glial activation and neuroinflammation, edema, ischemia, vascular injury, energy failure, and peripheral immune cell infiltration. The timing of these events post injury has been linked to injury severity and functional outcome. Extracellular vesicles are membrane bound secretory vesicles that contain markers and cargo pertaining to their cell of origin and can cross the blood-brain barrier. These qualities make extracellular vesicles intriguing candidates for a liquid biopsy into the pathophysiologic changes occurring at the cellular level post traumatic brain injury. Herein, we review the most commonly reported cargo changes in extracellular vesicles from clinical traumatic brain injury samples. We then use knowledge from animal and in vitro models to help infer what these changes may indicate regrading cellular responses post traumatic brain injury. Future research should prioritize labeling extracellular vesicles with markers for distinct cell types across a range of timepoints post traumatic brain injury.

Mesenchymal stem cell-derived extracellular vesicles in skin wound healing:roles,opportunities and challenges

Skin wounds are characterized by injury to the skin due to trauma,tearing,cuts,or contusions.As such injuries are common to all human groups,they may at times represent a serious socioeconomic burden.Currently,increasing numbers of studies have focused on the role of mesenchymal stem cell(MSC)-derived extracellular vesicles(EVs)in skin wound repair.As a cell-free therapy,MSC-derived EVs have shown significant application potential in the field of wound repair as a more stable and safer option than conventional cell therapy.Treatment based on MSC-derived EVs can significantly promote the repair of damaged substructures,including the regeneration of vessels,nerves,and hair follicles.In addition,MSC-derived EVs can inhibit scar formation by affecting angiogenesis-related and antifibrotic pathways in promoting macrophage polarization,wound angiogenesis,cell proliferation,and cell migration,and by inhibiting excessive extracellular matrix production.Additionally,these structures can serve as a scaffold for components used in wound repair,and they can be developed into bioengineered EVs to support trauma repair.Through the formulation of standardized culture,isolation,purification,and drug delivery strategies,exploration of the detailed mechanism of EVs will allow them to be used as clinical treatments for wound repair.In conclusion,MSCderived EV-based therapies have important application prospects in wound repair.Here we provide a comprehensive overview of their current status,application potential,and associated drawbacks.

Small extracellular vesicles from hypoxia-preconditioned bone marrow mesenchymal stem cells attenuate spinal cord injury via miR-146a-5p-mediated regulation of macrophage polarization

Spinal cord injury is a disabling condition with limited treatment options.Multiple studies have provided evidence suggesting that small extracellular vesicles(SEVs)secreted by bone marrow mesenchymal stem cells(MSCs)help mediate the beneficial effects conferred by MSC transplantation following spinal cord injury.Strikingly,hypoxia-preconditioned bone marrow mesenchymal stem cell-derived SEVs(HSEVs)exhibit increased therapeutic potency.We thus explored the role of HSEVs in macrophage immune regulation after spinal cord injury in rats and their significance in spinal cord repair.SEVs or HSEVs were isolated from bone marrow MSC supernatants by density gradient ultracentrifugation.HSEV administration to rats via tail vein injection after spinal cord injury reduced the lesion area and attenuated spinal cord inflammation.HSEVs regulate macrophage polarization towards the M2 phenotype in vivo and in vitro.Micro RNA sequencing and bioinformatics analyses of SEVs and HSEVs revealed that mi R-146a-5p is a potent mediator of macrophage polarization that targets interleukin-1 receptor-associated kinase 1.Reducing mi R-146a-5p expression in HSEVs partially attenuated macrophage polarization.Our data suggest that HSEVs attenuate spinal cord inflammation and injury in rats by transporting mi R-146a-5p,which alters macrophage polarization.This study provides new insights into the application of HSEVs as a therapeutic tool for spinal cord injury.

High quality repair of osteochondral defects in rats using the extracellular matrix of antler stem cells

BACKGROUND Cartilage defects are some of the most common causes of arthritis.Cartilage lesions caused by inflammation,trauma or degenerative disease normally result in osteochondral defects.Previous studies have shown that decellularized extracellular matrix(ECM)derived from autologous,allogenic,or xenogeneic mesenchymal stromal cells(MSCs)can effectively restore osteochondral integrity.AIM To determine whether the decellularized ECM of antler reserve mesenchymal cells(RMCs),a xenogeneic material from antler stem cells,is superior to the currently available treatments for osteochondral defects.METHODS We isolated the RMCs from a 60-d-old sika deer antler and cultured them in vitro to 70%confluence;50 mg/mL L-ascorbic acid was then added to the medium to stimulate ECM deposition.Decellularized sheets of adipocyte-derived MSCs(aMSCs)and antlerogenic periosteal cells(another type of antler stem cells)were used as the controls.Three weeks after ascorbic acid stimulation,the ECM sheets were harvested and applied to the osteochondral defects in rat knee joints.RESULTS The defects were successfully repaired by applying the ECM-sheets.The highest quality of repair was achieved in the RMC-ECM group both in vitro(including cell attachment and proliferation),and in vivo(including the simultaneous regeneration of well-vascularized subchondral bone and avascular articular hyaline cartilage integrated with surrounding native tissues).Notably,the antler-stem-cell-derived ECM(xenogeneic)performed better than the aMSC-ECM(allogenic),while the ECM of the active antler stem cells was superior to that of the quiescent antler stem cells.CONCLUSION Decellularized xenogeneic ECM derived from the antler stem cell,particularly the active form(RMC-ECM),can achieve high quality repair/reconstruction of osteochondral defects,suggesting that selection of decellularized ECM for such repair should be focused more on bioactivity rather than kinship.

MiRNA-145-5p inhibits gastric cancer progression via the serpin family E member 1-extracellular signal-regulated kinase-1/2 axis

BACKGROUND MicroRNAs(miRNAs)regulate gene expression and play a critical role in cancer physiology.However,there is still a limited understanding of the function and regulatory mechanism of miRNAs in gastric cancer(GC).AIM To investigate the role and molecular mechanism of miRNA-145-5p(miR145-5p)in the progression of GC.METHODS Real-time polymerase chain reaction(RT-PCR)was used to detect miRNA expression in human GC tissues and cells.The ability of cancer cells to migrate and invade was assessed using wound-healing and transwell assays,respectively.Cell proliferation was measured using cell counting kit-8 and colony formation assays,and apoptosis was evaluated using flow cytometry.Expression of the epithelial-mesenchymal transition(EMT)-associated protein was determined by Western blot.Targets of miR-145-5p were predicated using bioinformatics analysis and verified using a dual-luciferase reporter system.Serpin family E member 1(SERPINE1)expression in GC tissues and cells was evaluated using RT-PCR and immunohistochemical staining.The correlation between SERPINE1 expression and overall patient survival was determined using Kaplan-Meier plot analysis.The association between SERPINE1 and GC progression was also tested.A rescue experiment of SERPINE1 overexpression was conducted to verify the relationship between this protein and miR-145-5p.The mechanism by which miR-145-5p influences GC progression was further explored by assessing tumor formation in nude mice.RESULTS GC tissues and cells had reduced miR-145-5p expression and SERPINE1 was identified as a direct target of this miRNA.Overexpression of miR-145-5p was associated with decreased GC cell proliferation,invasion,migration,and EMT,and these effects were reversed by forcing SERPINE1 expression.Kaplan-Meier plot analysis revealed that patients with higher SERPINE1 expression had a shorter survival rate than those with lower SERPINE1 expression.Nude mouse tumorigenesis experiments confirmed that miR-145-5p targets SERPINE1 to regulate extracellular signal-regulated kinase-1/2(ERK1/2).CONCLUSION This study found that miR-145-5p inhibits tumor progression and is expressed in lower amounts in patients with GC.MiR-145-5p was found to affect GC cell proliferation,migration,and invasion by negatively regulating SERPINE1 levels and controlling the ERK1/2 pathway.

tRNA^(Glu)-derived fragments from embryonic extracellular vesicles modulate bovine embryo hatching

Transfer RNA-derived small RNAs(tsRNAs)have been shown to be involved in early embryo development and repression of endogenous retroelements in embryos and stem cells.However,it is unknown whether tsRNAs also regulate embryo hatching.In this study,we mined the sequencing data of a previous experiment in which we demonstrated that the microRNA(miRNA)cargo of preimplantation embryonic extracellular vesicles(EVs)influences embryo development.We thus profiled the tsRNA cargo of EVs secreted by blastocysts and non-blastocysts.The majority of tsRNAs was identified as tRNA halves originating from the 5'ends of tRNAs.Among the 148 differentially expressed tsRNAs,the 19 nt tRNA fragment(tRF)tDR-14:32-Glu-CTC-1 was found to be significantly up-regulated in EVs derived from non-blastocysts.RT-qPCR assays confirmed its significant up-regulation in non-blastocyst embryos and their conditioned medium compared to the blastocyst group(P<0.05).Inhibition of tDR-14:32-Glu-CTC-1 by supplementing antagomirs to the conditioned medium improved embryo hatching(P<0.05).Transcriptomic analysis of embryos treated with tDR-14:32-Glu-CTC-1 antagomirs further showed differential expression of genes that are associated with embryo hatching and implantation.In summary,tDR-14:32-Glu-CTC-1 is up-regulated in non-blastocyst embryos and their secretions,and inhibition of tDR-14:32-Glu-CTC-1 promotes embryo hatching,while influencing embryo implantation-related genes and pathways.These results indicate that embryonic EVs containing specific tRFs may regulate preimplantation embryo development.

Stiffness-tunable biomaterials provide a good extracellular matrix environment for axon growth and regeneration

Neuronal growth, extension, branching, and formation of neural networks are markedly influenced by the extracellular matrix—a complex network composed of proteins and carbohydrates secreted by cells. In addition to providing physical support for cells, the extracellular matrix also conveys critical mechanical stiffness cues. During the development of the nervous system, extracellular matrix stiffness plays a central role in guiding neuronal growth, particularly in the context of axonal extension, which is crucial for the formation of neural networks. In neural tissue engineering, manipulation of biomaterial stiffness is a promising strategy to provide a permissive environment for the repair and regeneration of injured nervous tissue. Recent research has fine-tuned synthetic biomaterials to fabricate scaffolds that closely replicate the stiffness profiles observed in the nervous system. In this review, we highlight the molecular mechanisms by which extracellular matrix stiffness regulates axonal growth and regeneration. We highlight the progress made in the development of stiffness-tunable biomaterials to emulate in vivo extracellular matrix environments, with an emphasis on their application in neural repair and regeneration, along with a discussion of the current limitations and future prospects. The exploration and optimization of the stiffness-tunable biomaterials has the potential to markedly advance the development of neural tissue engineering.

Biofabrication of nanocomposite-based scaffolds containing human bone extracellularmatrix for the differentiation of skeletal stem and progenitor cells

Autograft or metal implants are routinely used in skeletal repair.However,they fail to provide long-term clinical resolution,necessitating a functional biomimetic tissue engineering alternative.The use of native human bone tissue for synthesizing a biomimeticmaterial inkfor three-dimensional(3D)bioprintingof skeletal tissueis anattractivestrategyfor tissueregeneration.Thus,human bone extracellular matrix(bone-ECM)offers an exciting potential for the development of an appropriate microenvironment for human bone marrow stromal cells(HBMSCs)to proliferate and differentiate along the osteogenic lineage.In this study,we engineered a novel material ink(LAB)by blending human bone-ECM(B)with nanoclay(L,Laponite®)and alginate(A)polymers using extrusion-based deposition.The inclusion of the nanofiller and polymeric material increased the rheology,printability,and drug retention properties and,critically,the preservation of HBMSCs viability upon printing.The composite of human bone-ECM-based 3D constructs containing vascular endothelial growth factor(VEGF)enhanced vascularization after implantation in an ex vivo chick chorioallantoic membrane(CAM)model.The inclusion of bone morphogenetic protein-2(BMP-2)with the HBMSCs further enhanced vascularization and mineralization after only seven days.This study demonstrates the synergistic combination of nanoclay with biomimetic materials(alginate and bone-ECM)to support the formation of osteogenic tissue both in vitro and ex vivo and offers a promising novel 3D bioprinting approach to personalized skeletal tissue repair.

Utilizing engineered extracellular vesicles as delivery vectors in the management of ischemic stroke:a special outlook on mitochondrial delivery

Ischemic stroke is a secondary cause of mortality worldwide,imposing considerable medical and economic burdens on society.Extracellular vesicles,serving as natural nanocarriers for drug delivery,exhibit excellent biocompatibility in vivo and have significant advantages in the management of ischemic stroke.However,the uncertain distribution and rapid clearance of extracellular vesicles impede their delivery efficiency.By utilizing membrane decoration or by encapsulating therapeutic cargo within extracellular vesicles,their delivery efficacy may be greatly improved.Furthermore,previous studies have indicated that microvesicles,a subset of large-sized extracellular vesicles,can transport mitochondria to neighboring cells,thereby aiding in the restoration of mitochondrial function post-ischemic stroke.Small extracellular vesicles have also demonstrated the capability to transfer mitochondrial components,such as proteins or deoxyribonucleic acid,or their sub-components,for extracellular vesicle-based ischemic stroke therapy.In this review,we undertake a comparative analysis of the isolation techniques employed for extracellular vesicles and present an overview of the current dominant extracellular vesicle modification methodologies.Given the complex facets of treating ischemic stroke,we also delineate various extracellular vesicle modification approaches which are suited to different facets of the treatment process.Moreover,given the burgeoning interest in mitochondrial delivery,we delved into the feasibility and existing research findings on the transportation of mitochondrial fractions or intact mitochondria through small extracellular vesicles and microvesicles to offer a fresh perspective on ischemic stroke therapy.

Advances in extracellular vesicle-based combination therapies for spinal cord injury

Spinal cord injury is a severe insult to the central nervous system that causes persisting neurological deficits.The currently available treatments involve surgical,medical,and rehabilitative strategies.However,none of these techniques can markedly reverse neurological deficits.Recently,extracellular vesicles from various cell sources have been applied to different models of spinal cord injury,thereby generating new cell-free therapies for the treatment of spinal cord injury.However,the use of extracellular vesicles alone is still associated with some notable shortcomings,such as their uncertainty in targeting damaged spinal cord tissues and inability to provide structural support to damaged axons.Therefore,this paper reviews the latest combined strategies for the use of extracellular vesicle-based technology for spinal cord injury,including the combination of extracellular vesicles with nanoparticles,exogenous drugs and/or biological scaffold materials,which facilitate the targeting ability of extracellular vesicles and the combinatorial effects with extracellular vesicles.We also highlight issues relating to the clinical transformation of these extracellular vesicle-based combination strategies for the treatment of spinal cord injury.

Treatment of hemophilic arthropathy by immunomodulatory extracellular vesicle delivered by liposome hybrid nanoparticles

In individuals afflicted with hemophilia,characterized by a deficiency of coagulation factor VIII(FVIII),the occurrence of spontaneous recurrent intra-articular hemorrhage precipitates the emergence of hemophilic arthropathy(HA).Although clotting factor replacement therapy reduces joint bleeding clinically,clotting factors need to be injected frequently due to the rapid diffusion of the drug.Hence,a novel drug delivery approach may be developed to improve the drug therapy.Platelet-derived extracellular vesicles(PEVs)are known to possess anti-inflammatory and hemostatic properties and could be used as a potential HA therapy.In this study,we constructed a PEV-LS@FVIII nanotherapeutic system by combining thioketal(TK),liposomes(LS),and FVIII to form the LS@FVIII complexes,and then hybridizing PEV with LS@FVIII.Our results demonstrated that PEVLS@FVIII could efficiently facilitate FVIII delivery and specifically target the injured knee joint.Both in vitro and in vivo studies showed a reduction in the M1 phenotype of macrophages and an enhancement of the M2 phenotype,compared to FVIII free control.Furthermore,PEV-LS@FVIII appeared to alleviate HA-induced cartilage damage.In conclusion,our findings demonstrate that PEV-LS@FVIII could delay the progression of HA by targeting bleeding joints,modulating macrophage polarization to suppress inflammation,and mitigating cartilage damage.

Small extracellular vesicles derived from cerebral endothelial cells with elevated microRNA 27a promote ischemic stroke recovery

Axonal remodeling is a critical aspect of ischemic brain repair processes and contributes to spontaneous functional recovery.Our previous in vitro study demonstrated that exosomes/small extracellular vesicles(sEVs)isolated from cerebral endothelial cells(CEC-sEVs)of ischemic brain promote axonal growth of embryonic cortical neurons and that microRNA 27a(miR-27a)is an elevated miRNA in ischemic CEC-sEVs.In the present study,we investigated whether normal CEC-sEVs engineered to enrich their levels of miR-27a(27a-sEVs)further enhance axonal growth and improve neurological outcomes after ischemic stroke when compared with treatment with non-engineered CEC-sEVs.27a-sEVs were isolated from the conditioned medium of healthy mouse CECs transfected with a lentiviral miR-27a expression vector.Small EVs isolated from CECs transfected with a scramble vector(Scra-sEVs)were used as a control.Adult male mice were subjected to permanent middle cerebral artery occlusion and then were randomly treated with 27a-sEVs or Scra-sEVs.An array of behavior assays was used to measure neurological function.Compared with treatment of ischemic stroke with Scra-sEVs,treatment with 27a-sEVs significantly augmented axons and spines in the peri-infarct zone and in the corticospinal tract of the spinal grey matter of the denervated side,and significantly improved neurological outcomes.In vitro studies demonstrated that CEC-sEVs carrying reduced miR-27a abolished 27a-sEV-augmented axonal growth.Ultrastructural analysis revealed that 27a-sEVs systemically administered preferentially localized to the pre-synaptic active zone,while quantitative reverse transcription-polymerase chain reaction and Western Blot analysis showed elevated miR-27a,and reduced axonal inhibitory proteins Semaphorin 6A and Ras Homolog Family Member A in the peri-infarct zone.Blockage of the Clathrin-dependent endocytosis pathway substantially reduced neuronal internalization of 27a-sEVs.Our data provide evidence that 27a-sEVs have a therapeutic effect on stroke recovery by promoting axonal remodeling and improving neurological outcomes.Our findings also suggest that suppression of axonal inhibitory proteins such as Semaphorin 6A may contribute to the beneficial effect of 27a-sEVs on axonal remodeling.

Nano-vibration exciter:Hypoxia-inducible factor 1 signaling pathway-mediated extracellular vesicles as bioactive glass substitutes for bone regeneration

Bioactive glasses(BG)play a vital role in angiogenesis and osteogenesis through releasing functional ions.However,the rapid ion release in the early stage will cause excessive accumulation of metal ions,which in turn leads to obvious cytotoxicity,long-term inflammation,and bone repair failure.Inspired by the vibration exciter,small extracellular vesicles(sEVs)obtained by treating mesenchymal stem cells with copper-doped bioactive glass(CuBG-sEVs),is prepared as a nano-vibration exciter.The nano-vibration exciter can convert the ion signals of CuBG into biochemical factor signals through hypoxia-inducible factor 1(HIF-1)signaling pathway and its activated autophagy,so as to better exert the osteogenic activity of BG.The results showed that CuBG extracts could significantly improve the enrichment of key miRNAs and increase the yield of CuBG-sEVs by activating HIF-1 signaling pathway and its activated autophagy.Cell experiments showed that CuBG-sEVs are favor to cell recruitment,vascularization and osteogenesis as the enrichment of key miRNAs.The animal experiments results showed that CuBG-sEVs stimulated angiogenesis mediated by CD31 and promoted bone regeneration by activating signaling pathways related to osteogenesis.These findings underscored the significant potential of sEVs as alternative strategies to better roles of BG.

High throughput and rapid isolation of extracellular vesicles and exosomes with purity using size exclusion liquid chromatography

Extracellular vesicles(EVs)have emerged as potential biomarkers for diagnosing a range of diseases without invasive procedures.Extracellular vesicles also offer advantages compared to synthetic vesicles for delivery of various drugs;however,limitations in segregating EVs from other particles and soluble proteins have led to inconsistent EV retrieval rates with low levels of purity.Here,we report a new high-yield(88.47%)and rapid(<20 min)EV isolation method termed size exclusion–fast protein liquid chromatography(SE-FPLC).We show SE-FPLC can effectively isolate EVs from multiple sources including EVs derived from human and mouse cells and serum samples.The results indicate that SE-FPLC can successfully remove highly abundant protein contaminants such as albumin and lipoprotein complexes,which can represent a major hurdle in large scale isolation of EVs.The high-yield nature of SE-FPLC allows for easy industrial scaling up of EV production for various clinical utilities.SE-FPLC also enables analysis of small volumes of blood for use in point-of-care diagnostics in the clinic.Collectively,SE-FPLC offers many advantages over current EV isolation methods and offers rapid clinical translation.

Small extracellular vesicles derived from human induced pluripotent stem cell-differentiated neural progenitor cells mitigate retinal ganglion cell degeneration in a mouse model of optic nerve injury

Several studies have found that transplantation of neural progenitor cells(NPCs)promotes the survival of injured neurons.However,a poor integration rate and high risk of tumorigenicity after cell transplantation limits their clinical application.Small extracellular vesicles(sEVs)contain bioactive molecules for neuronal protection and regeneration.Previous studies have shown that stem/progenitor cell-derived sEVs can promote neuronal survival and recovery of neurological function in neurodegenerative eye diseases and other eye diseases.In this study,we intravitreally transplanted sEVs derived from human induced pluripotent stem cells(hiPSCs)and hiPSCs-differentiated NPCs(hiPSC-NPC)in a mouse model of optic nerve crush.Our results show that these intravitreally injected sEVs were ingested by retinal cells,especially those localized in the ganglion cell layer.Treatment with hiPSC-NPC-derived sEVs mitigated optic nerve crush-induced retinal ganglion cell degeneration,and regulated the retinal microenvironment by inhibiting excessive activation of microglia.Component analysis further revealed that hiPSC-NPC derived sEVs transported neuroprotective and anti-inflammatory miRNA cargos to target cells,which had protective effects on RGCs after optic nerve injury.These findings suggest that sEVs derived from hiPSC-NPC are a promising cell-free therapeutic strategy for optic neuropathy.

From the dust:extracellular vesicles as regulators of development and neuroregeneration

First impressions can have a lasting impact.In science,this can be especially problematic,because our lack of understanding often causes us to mislabel and thus ignore important biological processes.In this vein,extracellular vesicles(EVs)were once considered to be“cellular dust”.Similar to the previous concept of“junk DNA”to describe protein non-coding regions,EVs are far more than just cellular dust.In fact,EVs are emerging as key mediators of intracellular communication across nearly all biological systems.This includes peripheral immune responses(e.g.,arthropathies and sepsis),intra-organ communication(Seim et al.,2023),and a host of other physiological and pathological states(Figure 1A and B).