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Fabrication and characterization of PBP‐EVs. a) Landscape atlas of miRNAs within EVs derived from three different donors. b) Venn diagram of abundantly expressed miRNAs identified in EVs derived from three donors. c) Schematic illustration of PBP‐EV fabrication through anchoring PBP on the EV membrane by DMPE‐PEG. Cy5.5 was conjugated on PBP side chains to visualize the surface modification of DMPE‐PEG‐PBP (DPP). d) Flow cytometry to assess the modification of Cy5.5‐labeled DPP with different molar concentrations on EVs. e) Flow cytometry to assess the efficiency of Cy5.5‐labeled DPP modification on EVs after incubation for different times. f) Size distributions of EVs and PBP‐EVs. g) Zeta potentials of EVs and PBP‐EVs, n = 3. ns, not significant. Statistical analysis was performed using a two‐tailed unpaired Student's t‐test. h) Transmission electron microscopy (TEM) images of EVs and PBP‐EVs. Scale bar, 100 nm. i) Western blot analysis confirmed the three categories of EV markers (ALIX, TSG101, and CD63) in EVs and PBP‐EVs. The hP‐MSC lysate served as a control. j) Flow cytometry to evaluate the stability of PBP‐EVs preserved at 4 °C for 1, 3, and 7 days.
Source publication
Endothelial cell injury plays a critical part in ischemic acute kidney injury (AKI) and participates in the progression of AKI. Targeting renal endothelial cell therapy may ameliorate vascular injury and further improve the prognosis of ischemic AKI. Here, P-selectin as a biomarker of ischemic AKI in endothelial cells is identified and P-selectin b...
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Citations
... Following tissue injury, VECs can quickly up-regulate P-selectin, a cell adhesion molecule, which mediates monocyte slow rolling, arrest, and migration [105]. Zhang et al. found that P-selectin binding peptide-modified human placenta mesenchymal stem cell-derived extracellular vesicles (MSC-EVs) specially target damaged endothelial cells, reduce moMϕ adhesion and promote kidney reparative angiogenesis to assist kidney repair [94]. ...
Acute kidney injury (AKI), triggered by ischemia, sepsis, toxicity, or obstruction, is marked by a rapid impairment of renal function and could lead to the initiation and advancement of chronic kidney disease (CKD). The concept of AKI to CKD transition has gained much interest. Despite a series of studies highlighting the diverse roles of renal macrophages in the immune response following AKI, the intricate mechanisms of macrophage-driven cell–cell communication in AKI to CKD transition remains incompletely understood. In this review, we introduce the dynamic phenotype change of macrophages under the different stages of kidney injury. Importantly, we present novel perspectives on the extensive interaction of renal macrophages with adjacent cells, including tubular epithelial cells, vascular endothelial cells, fibroblasts, and other immune cells via soluble factors, extracellular vesicles, and direct contact, to facilitate the transition from AKI to CKD. Additionally, we summarize the potential therapeutic strategies based on the adverse macrophage-neighboring cell crosstalk.
... Diabetic kidney disease (DKD), the most severe microvascular complication of diabetes, is the leading cause of end-stage renal disease (ESRD) and is related to markedly increased morbidity and mortality of cardiovascular disease worldwide [1][2][3]. The prominent pathological manifestations of DKD are glomerular hypertrophy, thickening of the basement membrane and mesangial expansion, which are caused by mesangial cell remolding, eventually leading to glomerulosclerosis [4]. ...
... Recent studies have indicated that the therapeutic potential of MSCs relies mainly on EVs secreted by MSCs [24,25]. Extracellular vesicles (EVs), which are membrane-packed nanovesicles, mediate intercellular communication by transferring cargos (miRNAs, circular RNAs, proteins and lipids) to target cells and are a cell-free therapy alternative, with the advantages of preventing immunogenicity, tumorigenicity, easy storage and production, and passing through biological barriers [3,26,27]. Studies have shown that MSC-EVs can yield beneficial therapeutic effects on DKD [28,29]. However, the underlying mechanisms by which MSC-EVs alleviate DKD remain to be fully elucidated. ...
... EVs were harvested from the supernatant of hP-MSCs as previously described [3,27]. Briefly, EV-free FBS was obtained by centrifugation of FBS at 100,000 × g for 2 h. ...
Background
Diabetic kidney disease (DKD) is the leading cause of end-stage renal disease (ESRD) globally, presenting a significant therapeutic challenge. Extracellular vesicles (EVs) from mesenchymal stem cells (MSCs) have emerged as promising therapeutic agents. This study explored the therapeutic effects and mechanisms of EVs derived from human placental mesenchymal stem cells (hP-MSCs) on DKD.
Methods
EVs were isolated from cultured hP-MSCs and administered to streptozotocin (STZ)-induced diabetic mice and high glucose–treated glomerular mesangial cells. The therapeutic impact of EVs was assessed through histological analysis and biochemical assays. miR-99b-5p expression in EVs and its role in modulating the mechanistic target of rapamycin (mTOR)/autophagy pathway were examined via western blotting and RT‒qPCR.
Results
Treatment with hP-MSC-derived EVs significantly alleviated renal fibrosis and improved renal function in DKD models. These EVs were enriched with miR-99b-5p, which targeted and inhibited mTOR signaling, thereby increasing autophagic activity and reducing cellular proliferation and extracellular matrix accumulation in renal tissues.
Conclusions
hP-MSC-derived EVs can mitigate renal injury in DKD by modulating the miR-99b-5p/mTOR/autophagy pathway. These findings suggest a potential cell-free therapeutic strategy for managing DKD.
... However, the mechanisms by which EVs reach these vessels remain unexplored. To facilitate EV targeting of endothelial cells, Zhang et al. (40) demonstrated that placenta-derived EVs engineered to express P-selectin-binding peptide showed better functionality in reducing inflammation, loss of capillaries, and maladaptive repair of tubules than those treated with non-engineered EVs on day 3 of IR injury. Furthermore, these engineered EVs showed enhanced anti-fibrotic functioning relative to non-engineered EVs on day 28 of injury, indicating that a single administration of EVs targeting the renal endothelium is sufficient to inhibit fibrosis (40). ...
... To facilitate EV targeting of endothelial cells, Zhang et al. (40) demonstrated that placenta-derived EVs engineered to express P-selectin-binding peptide showed better functionality in reducing inflammation, loss of capillaries, and maladaptive repair of tubules than those treated with non-engineered EVs on day 3 of IR injury. Furthermore, these engineered EVs showed enhanced anti-fibrotic functioning relative to non-engineered EVs on day 28 of injury, indicating that a single administration of EVs targeting the renal endothelium is sufficient to inhibit fibrosis (40). Combined with drug loading, targeting TECs or the endothelium would be next platform to enhance the therapeutic efficacy of EVs in renal diseases. ...
Compared with conventional mesenchymal stem cells (MSCs), induced mesenchymal stem cells (iMSCs) from induced pluripotent stem cells are unique cell sources for tissue regeneration. The effect of extracellular vesicles (EVs) secreted from iMSCs on inhibiting acute kidney injury (AKI) to chronic kidney disease (CKD) transition was not reported. In this study, we investigated whether EVs from iMSCs (iMSC-EVs) could inhibit AKI-to-CKD transition. iMSC-EVs exhibited the general characteristics of EVs, such as protein marker expression, morphology, and size. Additionally, iMSC-EVs were detected in renal tissues after intravenous injection. In human renal tubular epithelial cells, the increase in pro-fibrotic gene expression in response to transforming growth factor β1 treatment was decreased by iMSC-EVs. In a mouse model of the AKI-to-CKD transtion induced by folic acid, repeated administration of iMSC-EVs restored renal function at day 14. Specifically, iMSC-EVs reduced interstitial fibrosis, sustained inflammation, various types of cell death, and the number of immune cells infiltrating kidneys. Capillary rarefaction in renal tissue was also reversed by iMSC-EVs. Our results demonstrate that iMSC-EVs reduced interstitial fibrosis, inflammation, and cell death occurring during the CKD transition after AKI. Thus, iMSC-EVs have the potential to block AKI-to-CKD transition.
... Among these, P-selectin is an inflammatory cell adhesion molecule. In the early phase of renal IRI, P-selectin is expressed in the glomeruli and interstitial capillaries of the injured kidney, mediating the adhesion of leukocytes to the inner walls of the damaged vessels and their migration to the injured cortex, thereby promoting intrarenal sterile inflammation [22]. Therefore, targeting and repairing damaged endothelial cells is of critical importance for improving the immune microenvironment. ...
... Based on this background, our research group has previously engineered EVs using P-selectin binding peptide (PBP) to create PBP-EVs, which target and repair endothelial cells in IRI kidneys [22]. Additionally, we used transcriptomic sequencing to observe changes in inflammation-related genes in the kidneys of AKI mice before and after treatment. ...
... Based on the foundation of previous laboratory research [22], we designed PBP-EVs that can target the P-selectin which on surface of damaged endothelial cells (Fig. 1A). The peptide fragment (DAEWVDVS) serves as a P-selectin binding peptide (PBP), enabling specific targeting and binding to P-selectin (Fig. S1a). ...
Background
Acute kidney injury (AKI) is a common and severe clinical condition. However, the underlying mechanisms of AKI have not been fully elucidated, and effective treatment options remain limited. Studies have shown that immune cells play a critical role in AKI, with regulatory T cells (Tregs) being one of the most important immunosuppressive lymphocytes. Tregs proliferation can attenuate AKI, whereas depletion exacerbates kidney injury. Given that endothelial cells (ECs) are the initial cells that interact with immune cells when they invade the tissue parenchyma, ECs are closely associated with immune reactions.
Methods and results
In this study, P-selectin binding peptide-extracellular vesicles (PBP-EVs) that target and repair ECs are engineered. Transcriptome sequencing reveals that PBP-EVs reduce the expression of inflammatory genes in AKI mice. Using high-resolution intravital two-photon microscopy (TPM), an increased recruitment of Tregs in the kidneys of AKI Foxp3-EGFP transgenic mice following PBP-EVs treatment is observed, as well as significant Lgr5⁺ renal stem cell proliferation in AKI Lgr5-CreERT2; R26mTmG mice. Additionally, PBP-EVs treatment result in reduced infiltration of inflammatory cells, pathological damage and fibrosis of AKI mice. Upon depletion of Tregs in Foxp3-DTR transgenic mice, we observe diminished therapeutic effect of PBP-EVs on AKI.
Conclusions
The experimental results indicate that PBP-EVs can promote the repair and regeneration of AKI by mitigating endothelial cell damage and subsequently modulating Tregs and the immune microenvironment. These findings provide novel insights and strategies for the treatment of AKI.
... Recent studies have demonstrated that the targeting ability of EVs can be improved through surface modification with specific cell-or tissue-targeting moieties. [42][43][44] One commonly used approach involves genetic modification, where parent cells are engineered to secrete functionalized EVs. 45,46 However, these processes are complex, time-intensive, and less suitable for difficult-to-transfect cell types, such as primary cells and stem cells. ...
... Consequently, EVs hold considerable potential as siRNA delivery systems to treat cardiac injuries. [42][43][44] However, unmodified EVs face notable limitations when administered systemically, such as non-specific accumulation in tissues, especially in the liver, and rapid clearance by macrophages. 40,41 To overcome these challenges, we modified hEV with the FAP aptamer to enable selective targeting of FAP, which is abundantly expressed in injured cardiac tissues and serves as an ideal target for site-specific delivery. ...
Purpose
Small‐interfering RNA (siRNA) therapy holds significant potential for treating cardiac injury; however, its clinical application is constrained by poor blood stability and insufficient cellular uptake. Extracellular vesicles (EVs) have emerged as an effective delivery system for siRNA in vivo; but their lack of specific cell or tissue‐targeting ability remains a major challenge. Thus, we aimed to develop an EV-based delivery system capable of targeted delivery of therapeutic siRNA to injured cardiac tissue for cardiac repair.
Methods
To identify fibroblast activation protein (FAP) as a potential target for delivery to injured cardiac tissue, we analyzed cardiac tissues from patients with heart failure and angiotensin II (Ang II)-treated mice. Injured cardiac tissue-targeting EVs were developed by embedding a cholesterol-conjugated FAP aptamer, which specifically targets FAP, onto human serum-derived EVs (hEV).
Results
Our findings revealed that FAP is upregulated after cardiac injury, highlighting its potential as a target for siRNA delivery to injured cardiac tissues. We successfully developed FAP aptamer-functionalized hEV (hEV@FAP) and confirmed their typical EV characteristics, including morphology, size distribution, zeta potential, and marker protein expression. In addition, hEV@FAP demonstrated high targeting selectivity to FAP-positive regions both in vitro and in vivo. To treat cardiac injury, hEV@FAP were loaded with TGFβ1 siRNA (siTGFβ1), identified as a molecular target for cardiac repair. In Ang II‐treated mice, intravenous administration of hEV@FAP-siTGFβ1 effectively reduced Ang II‐induced TGFβ1 expression in cardiac tissues, attributed to the protective and targeting capabilities of hEV@FAP. Consequently, hEV@FAP-siTGFβ1 significantly improved cardiac function, reduced myocardial fibrosis, and decreased cardiomyocyte cross‐sectional area (P < 0.05) without inducing systemic toxicity.
Conclusion
hEV@FAP represents a novel approach for targeted delivery of therapeutic siRNA to injured cardiac tissues, providing a promising nanomedicine for cardiac repair.
... Ultimately, theranostic aims to attain precise control of therapy and dose (2). The combination of diagnostics and therapeutics has opened the doors for precise diagnosis and personalized treatments in many diseases such as cancer (5)(6)(7), vascular diseases (8)(9)(10), cartilage diseases (11)(12)(13), kidney diseases (14)(15)(16), Alzheimer's disease (17)(18)(19) and bacterial infections (20)(21)(22). ...
Nanotechnology has emerged as a highly promising platform for theranostics, offering dual capabilities in targeted imaging and therapy. Interactions between the nanomaterial and biological components determine the in vivo fate of these materials which makes the control of their surface properties of utmost importance. Nanoparticles with neutral or negative surface charge have a longer circulation time while positively charged nanoparticles have higher affinity to cells and better cellular uptake. This trade-off presents a key challenge in optimizing surface charge for theranostic applications. A sophisticated solution is an on-demand switch of surface charge, enabled by leveraging the distinct pH conditions at the target site. In this review, we explore the intricate relationship between pH and charge modulation, summarizing recent advances in pH-induced charge-switchable nanomaterials for theranostics over the past five years. Additionally, we discuss how these innovations enhance targeted drug delivery and imaging contrast and provide perspectives on future directions for this transformative field.
... The increase in P-selectin and VCAM-1 expression in RIRI was less pronounced after 24 h when compared to CKD after 5 days. Specifically, the upregulation was approximately six times higher for P-selectin and twelve times higher for VCAM-1 in RIRI [37][38][39], which ist hree times lower relative to their up-regulation in CKD. This might explain the beneficial effects of P-Esbp observed in RIRI but not in CKD. ...
Background/Objectives: Leukocytes play a significant role in both acute kidney injury (AKI) and chronic kidney disease (CKD), contributing to pathogenesis and tissue damage. The process of leukocyte infiltration into the inflamed tissues is mediated by the interactions between the leukocytes and cell adhesion molecules (CAMs, i.e., E-selectin, P-selectin, and VCAM-1) present on the inner surface of the inflamed vasculature. Directly interfering with these interactions is a viable strategy to limit the extent of excessive inflammation; however, several small-molecule drug candidates failed during clinical translation. We hypothesized that a synthetic polymer presenting multiple copies of the high-affinity E-selecting binding peptide (P-Esbp) could block E-selectin-mediated functions and decrease leukocytes infiltration, thus reducing the extent of inflammatory kidney injury. Methods: P-Esbp was synthesized by conjugating E-selecting binding peptide (Esbp) to N-(2-hydroxypropyl)methacrylamide (HPMA) copolymer with reactive ester groups via aminolysis. The effects of P-Esbp treatment on kidney injury were investigated in two different models: AKI model (renal ischemia—reperfusion injury—RIRI) and CKD model (adenine-induced kidney injury). Results: We found that the mRNA levels of E-selectin were up-regulated in the kidney following acute and chronic tissue injury. P-Esbp demonstrated an extended half-life time in the bloodstream, and the polymer accumulated significantly in the liver, lungs, and kidneys within 4 h post injection. Treatment with P-Esbp suppressed the up-regulation of E-selectin in mice with RIRI and attenuated the inflammatory process. In the adenine-induced CKD model, the use of the E-selectin blocking copolymer had little impact on the progression of kidney injury, owing to the compensating function of P-selectin and VCAM-1. Conclusion: Our findings provide valuable insights into the interconnection between CAMs and compensatory mechanisms in controlling leukocyte migration in AKI and CKD. The combination of multiple CAM blockers, given simultaneously, may provide protective effects for preventing excessive leukocyte infiltration and control renal injury.
... Transcriptomics is used to examine the RNA content, identify genetic material that can be transferred between cells, and influence gene expression and cellular behavior. Research by Zhang et al. revealed that EVs derived from MSCs contain therapeutic miRNA components that may provide a nephroprotective effect (49). Furthermore, a study identifies two distinct extracellular RNA (exRNA) profiles released by mast cells, termed high-density (HD) and low-density (LD) exRNA. ...
Extracellular vesicles (EVs) are nanosized particles secreted by cells that play crucial roles in intercellular communication, especially in the context of cardiovascular diseases (CVDs). These vesicles carry complex cargo, including proteins, lipids, and nucleic acids, that reflects the physiological or pathological state of their cells of origin. Multiomics analysis of cell-derived EVs has provided valuable insights into the molecular mechanisms underlying CVDs by identifying specific proteins and EV-bound targets involved in disease progression. Recent studies have demonstrated that engineered EVs, which are designed to carry specific therapeutic molecules or modified to enhance their targeting capabilities, hold promise for treating CVDs. Analysis of the EV proteome has been instrumental in identifying key proteins that can be targeted or modulated within these engineered vesicles. For example, proteins involved in inflammation, thrombosis, and cardiac remodeling have been identified as potential therapeutic targets. Furthermore, the engineering of EVs to increase their delivery to specific tissues, such as the myocardium, or to modulate their immunogenicity and therapeutic efficacy is an emerging area of research. By leveraging the insights gained from multiomics analyses, researchers are developing EV-based therapies that can selectively target pathological processes in CVDs, offering a novel and potentially more effective treatment strategy. This review integrates the core findings from EV multiomics analysis in the context of CVDs and highlights the potential of engineered EVs in therapeutic applications.
... medicine with similar therapeutic potential compared to their donor cells [1,2]. EVs, derived from stem cells, including embryonic stem (ES) cells, induced pluripotent stem (iPS) cells, and mesenchymal stem cells (MSCs), have been investigated in clinical for a variety of diseases, including COVID-19, lung diseases [3]. ...
... The ability of EVs to convey bioactive molecules across biological barriers makes them promising candidates for therapeutic intervention. Engineered EVs loaded with specific contents can be designed to target particular cell types or deliver therapeutic drugs [2]. This targeted approach has shown its potential for the treatment of various diseases, including acute kidney injury ( Fig. 1.2), neurodegenerative disorders, cardiovascular diseases, and inflammatory conditions. ...
With their ability to self-renewal and differentiation, stem cells have been widely applied for regenerative therapy. However, ethical concerns, potential tumorigenesis, and immunogenicity of stem cells limit their clinical application. Extracellular vesicles (EVs), especially derived from stem cells, contain the functional compositions from stem cells that have been widely employed in tissue repair and regenerative medicine, offering an alternative to stem cells for regenerative therapy. Recent studies indicate that EVs exhibit similar therapeutic effects compared to their donor cells, while minimizing the drawback associated with stem cell therapy. Unlike their cell counterparts, EVs are stable enough for long-term storage and can be easily engineered and modified for drug loading and targeting therapy. In this book, we will discuss recent advances in EV secretion, cargo packages, and translational application, and emphasize the multifaceted roles of EVs in regenerative therapy.
... Additionally, visualization of EV treatments can indicate the location and severity of tissue injury in vivo, integrating diagnosis and treatment into a single process. 98 Zhang, K. et al. reported a Cy5 and P-selectin binding peptide modified EVs, which can target bind to ischemia induced P-selectin expression on renal endothelial cells, could indicate the kidney injury degree and achieve the target treatment at the same time. 98 The combination of EV-based treatment and imaging technologies holds great promise for developing precise and personalized therapeutic strategies for tissue regeneration. ...
... 98 Zhang, K. et al. reported a Cy5 and P-selectin binding peptide modified EVs, which can target bind to ischemia induced P-selectin expression on renal endothelial cells, could indicate the kidney injury degree and achieve the target treatment at the same time. 98 The combination of EV-based treatment and imaging technologies holds great promise for developing precise and personalized therapeutic strategies for tissue regeneration. Currently, ongoing research includes real-time tracking of EVs in liver regeneration, as reported by Cao, H. et al. ...
