There is currently increasing focus on aging-related diseases. Osteoporosis is a common disease the incidence of which increases with age. In older patients with osteoporosis, bone marrow mesenchymal stem cells (BMMSCs) have a decreased capacity for osteogenesis and an increased capacity for adipogenesis, causing excessive accumulation of adipose tissue in the bone marrow. Therefore, means of reducing bone marrow adiposity may have therapeutic potential for osteoporosis. Apoptotic vesicles (apoVs) participate in a wide range of physiological processes and have been shown to have therapeutic effects in a variety of diseases. The principal objective of this study was to examine the special properties and regulatory mechanisms of BMMSC-derived apoVs in the treatment of bone marrow adiposity.

The results showed that apoVs could decrease bone marrow adiposity in osteoporotic mice and prevent adipogenic differentiation of MSCs by activating the Wnt/β-catenin pathway.

New apoV-based therapies have potential for the treatment of bone marrow adiposity in patients with aging-related osteoporosis and may be further applicable to the treatment of obesity and aging-related diseases.

Cell-membrane proteins are critical mediators of signal transduction, playing essential roles in disease occurrence and progression. The emerging LYTACs (Lysosome-targeting chimeras) technology combines drug-targeting strategies with lysosomal degradation, providing a novel approach to drug development and offering new possibilities for disease therapy. However, the clinical applicability of current LYTAC degraders is limited by the variable expression of lysosome-targeting receptors (LTRs) in tissues. To overcome this limitation, we herein hijacked a YXXØ sorting signal that derived from lysosome-associated membrane protein 2a (LAMP-2a) to develop a signal aptamer platform (SApt), which exhibits high specificity for targeting membrane proteins and inducing efficient lysosomal degradation. SApts were synthesized by conjugating the YXXØ signal peptide to the aptamer's terminus through a click reaction. Our study demonstrated that SApts efficiently degrade disease-associated membrane proteins, such as PTK7, Met, and NCL, based on the inherent signals rather than specific LTR. The potent antitumor efficacy of SApts was further confirmed in a xenograft tumor model, where in vivo degradation of PTK7 was observed. Collectively, our work provides insights into the development of a simple and universal lysosomal degradation platform with potential translational value in clinical treatment.

In recent decades, porous titanium (Ti) bone-engineered scaffolds have emerged as a promising biomaterial for bone defect repair due to their excellent biocompatibility and mechanical properties. However, the limited bioactivity on the surface of the porous scaffold hinders osteogenesis and osseointegration, thereby restricting its further application. In this study, we utilized surface-initiated atom transfer radical polymerization to prepare a zwitterionic poly[2-(methacryloyloxy)ethyl choline phosphate] (PMCP) modified bioactive coating on the surface of a 3D-printed porous Ti scaffold. Additionally, exosomes derived from bone mesenchymal stem cells (BMSCs) were introduced into the scaffold surface via specific interactions between choline phosphate and phosphatidylcholine (CP-PC) on exosomes. In vitro studies for ossification and transcriptome analysis have shown that the exosome bioactive coating on a Ti scaffold enhances the proliferation of BMSCs, their osteogenic activity, and the expression of osteogenic-related genes. Furthermore, in vivo study results from hard tissue sectioning and microcomputed tomography indicate that the bioactive Ti scaffold significantly promotes new bone formation after 4 and 12 weeks of implantation in rabbit femoral defects. Overall, this study showcases the potential of the exosome-based Ti scaffold to enhance osteogenic activity, offering a novel strategy for cell-free bone tissue regeneration with significant therapeutic implications.

Although immunotherapy demonstrates considerable prospect in overcoming solid tumors, its clinical efficacy is limited by several factors, such as poor tumor immunogenicity, inadequate immune activation, and immunosuppressive tumor microenvironment (TME). To overcome these challenges, a versatile and universal immune modulation platform should be developed, and lipid nanovesicles with membrane fusion capabilities (LNV-Fs) have attracted great attention for this purpose. By mimicking natural membrane fusion processes, LNV-Fs enable the precise presentation of immunogenic components on tumor cell membranes, effectively activating anti-tumor immune surveillance. Similarly, LNV-Fs can equip multiple functionalities on autologous and adoptive effector cells for enhanced cell therapies. Additionally, LNV-Fs function as vaccines that elicit robust autologous anti-tumor immunity while promoting long-term immune memory. Furthermore, different LNV-Fs with powerful ability in reprogramming TME have been reported. Given the recent advancements and the absence of comprehensive reviews on this topic, a comprehensive analysis of LNV-F systems, including their structural classifications, membrane fusion mechanisms, and recent applications in cancer immunotherapy is provided. Furthermore, the future prospects of LNV-Fs, with particular emphasis on artificial intelligence-assisted design are explored. This review is intended to engage researchers from diverse interdisciplinary fields and provide valuable insights for advancing precision immunotherapy.

Peptide assembly on the cell membrane is capable of endowing cells with novel biological properties that are distinct from their original states, thereby playing a pivotal role in the regulation of diverse cellular biological events. In practical biomedical scenarios, in order to make peptide assembly more precisely meet the requirements of cells at different physiological stages and conditions to achieve desired effects of cell function regulation, it becomes particularly crucial to conduct precise in situ spatiotemporal control of peptide assembly on the cell membrane, thus attracting great attentions. Particularly for tumor treatment, this artificially manipulated cell surface engineering can achieve excellent anti-tumor effects by altering the cell membrane structure, influencing receptor clustering or interfering with relevant signal pathways. Of note, membrane-anchoring peptides play a key role in these processes. In this review, we focus on three main types of membrane-anchoring peptides, elaborating in detail on how their assembly regulation mechanisms influence the cell membrane remodeling effect, and further exert therapeutic effects on tumors. On this basis, we further introduce a variety of tumor treatment strategies combined with in situ peptide assembly on the cell membrane, and discuss the current opportunities and challenges in this field, aiming to present the overall research panorama and trend of in situ peptide self-assembly on the cell membrane for efficient tumor treatment.

The p53 tumor suppressor gene, a master regulator of diverse cellular pathways, is frequently altered in various cancers. Loss of function in tumor suppressor genes is commonly associated with the onset/progression of cancer and treatment resistance. Currently, approaches for restoration of TP53 expression, including small molecules and DNA therapies, have yielded progressive success, but each has formidable drawbacks. Here, we introduced an endogenous nanoplatform to effectively deliver the TP53 protein. Briefly speaking, the endogenous TP53 proteins were fused by the Lamp2b and loaded into extracellular vesicles-based nanoparticles, which could markedly restore the TP53 expression in natural TP53-deficient ovarian cancer (OCs) and subsequently inhibit cell proliferation as well as induce cell apoptosis. Moreover, a well-known biotin streptavidin binding strategy was used to confer the nanoplatform targeting ability. Since mesothelin (MSLN) expressed highly in ovarian cancer, the anti-MSLN nanoplatform were engineered to deliver TP53 proteins to MSLN ovarian cancer and exert the anti-tumor ability. Our findings indicated that restoration of tumor suppressors by the targeting nanoplatform could be promising nanotechnology approaches for potential ovarian cancer treatment.

Intercellular communication is an essential hallmark of multicellular organisms for their development and adult tissue homeostasis. Over the past two decades, attention has been focused on communication mechanisms based on various membrane structures, as illustrated by the burst of scientific literature in the field of extracellular vesicles (EVs). These lipid bilayer-bound nano- or microparticles, as vehicle-like devices, act as regulators in various biological and physiological processes. When EVs are internalized by recipient cells, their membrane and cytoplasmic cargoes can interfere with cellular activities, affecting pathways that regulate cell proliferation, differentiation, and migration. In cancer, EVs can transfer oncogenic factors, stimulate neo-angiogenesis and immunosuppression, reprogram stromal cells, and confer drug resistance traits, thereby remodeling the surrounding microenvironment. Although the mechanisms underlying EV biogenesis and uptake are now better understood, little is known about the spatiotemporal mechanism(s) of their actions after internalization. In this respect, we have shown that a fraction of endocytosed EVs reaches the nuclear compartment via the VOR (VAP-A-ORP3-Rab7) complex-mediated docking of late endosomes to the outer nuclear membrane in the nucleoplasmic reticulum, positioning and facilitating the transfer of EV cargoes into the nucleoplasm via nuclear pores. Here, we highlight the EV heterogeneity, the cellular pathways governing EV release and uptake by donor and recipient cells, respectively, and focus on a novel intracellular pathway leading to the nuclear transfer of EV cargoes. We will discuss how to intercept it, which could open up new avenues for clinical applications in which EVs and other small extracellular particles (e.g., retroviruses) are implicated.

Enterovirus 71 (EV71) is a major etiologic pathogen for hand-foot-and-mouth disease (HFMD) in young children. Severe cases of EV71 infection could lead to neurological complications and even death, while the mechanism inducing neurological complications remains poorly understood. In this study, we firstly proved that microvesicles (MVs) could carry EV71 virions and mediate a higher efficiency in infection. Utilizing an in vitro blood-brain barrier (BBB) model, we observed that MVs containing virions (MVsEV71) could cross the BBB with greater efficiency compared to EV71 alone. Through in vivo imaging, we confirmed the ability of MVs to cross the BBB. qPCR assays showed a higher copy number of EV71 in both blood and brain samples in the mice treated with MVsEV71 compared to those treated with free EV71. Also, our investigation unveiled that MVsEV71 infection of animals induced cerebral hemorrhage and more severe inflammatory infiltration in the brain compared to animals infected by EV71 in vivo. Furthermore, we found a reduction in the expression of junction proteins such as zonula occludens-1 (ZO-1) and occludin. Moreover, the uptake of MVs by brain cells was examined using chemical inhibitor to block the endocytic pathway. Our experiments elucidated that the internalization of MVs occurred via a non-clathrin-dependent mechanism and a portion of the internalized MVs proceeded to enter lysosomes. In addition, we identified damaged mitochondria as the "cargo" of MVs, which facilitated MVsEV71 crossing the BBB and inducing cellular apoptosis. Meanwhile, MVsEV71 crossing the BBB further induced mitochondrial damaged and activated NOX4-derived ROS pathway in U251 cells. Taken together, these findings suggested that MVs transported EV71 virions across the BBB, while damaged mitochondria facilitated this process and aggravated the brain injury. Overall, these observations provide new insights into EV71-induced neurogenic complications and present a novel therapeutic target for the treatment of viral encephalitis.

Giardia intestinalis, a protozoan causing giardiasis, disrupts gastrointestinal health through complex host-parasite interactions. This study explores the differential uptake mechanisms of extracellular vesicles (EVs) derived from Giardia (gEVs), host cells (hEVs), and the host-parasite interaction (intEVs) in intestinal Caco-2 cells. Results show that intEVs are internalized more rapidly than gEVs and hEVs, underscoring their pivotal role in pathogenesis. To delineate uptake pathways, various endocytosis inhibitors were applied, and clathrin-mediated endocytosis inhibition using monodansylcadaverine (MDC) significantly reduced intEV and gEV uptake, confirming the role of clathrin-mediated endocytosis (CME). The use of dynasore, a dynamin inhibitor, strongly reduced the internalization of all EV types, demonstrating that uptake is dynamin-dependent. In contrast, methyl-β-cyclodextrin (MβCD), which disrupts lipid rafts and caveolae-mediated pathways, had no effect on EV uptake, indicating that caveolae are not involved in this process. Furthermore, inhibition of Na+/H+ exchange and phosphoinositide 3-kinase activity, both essential for macropinocytosis, also led to a significant reduction in intEV internalization. These findings strongly support that gEVs are internalized primarily through a dynamin- and clathrin-dependent pathway, independent of caveolae and lipid rafts, but modulated by tyrosine kinase signaling and macropinocytosis. These insights into selective and comprehensive inhibition pathways offer promising therapeutic targets to mitigate giardiasis.

Exosomes are microscopic vesicles released by cells that transport various biological materials and play a vital role in intercellular communication. When they are engineered, they serve as efficient delivery systems for therapeutic agents, making it possible to precisely deliver active pharmaceutical ingredients to organs, tissues, and cells. Exosomes' pharmacokinetics, or how they are transported and metabolized inside the body, is affected by several factors, including their source of origination and the proteins in their cell membranes. The pharmacokinetics and mobility of both native and modified exosomes are being observed in living organisms using advanced imaging modalities such as in vitro-in vivo simulation, magnetic resonance imaging, and positron emission tomography. Establishing comprehensive criteria for the investigation of exosomal pharmacokinetic is essential, given its increasing significance in both therapy and diagnostics. To obtain a thorough understanding of exosome intake, distribution, metabolism, and excretion, molecular imaging methods are crucial. The development of industrial processes and therapeutic applications depends on the precise measurement of exosome concentration in biological samples. To ensure a seamless incorporation of exosomes into clinical practice, as their role in therapeutics grows, it is imperative to conduct a complete assessment of their pharmacokinetics. This review provides a brief on how exosome-based research is evolving and the need for pharmacokinetic consideration to realize the full potential of these promising new therapeutic approaches.

Pore-forming peptides are promising antimicrobial and anticancer agents due to their membrane selectivity and biodegradability. Our prior work identified peptide M159, which permeabilized synthetic phosphatidylcholine liposomes without mammalian cell toxicity. Here, we report that the D-type variant (D-M159) induces apoptosis in HeLa cells under starvation. To explore its anticancer mechanism, we analyzed D-M159 cytotoxicity, intracellular uptake, and apoptotic pathways via flow cytometry, confocal microscopy, and Western blot. Calcium dynamics and mitochondrial function were examined via specific labeling and functional assays. Results revealed that D-M159 exhibited starvation-dependent, dose-responsive cytotoxicity and triggered apoptosis in HeLa cells. Mechanistic studies indicated that D-M159 entered the cells via caveolin-dependent and caveolae-dependent endocytosis pathways and induced endoplasmic reticulum stress in HeLa cells by up-regulating proteins such as ATF6, p-IRE1, PERK, GRP78, and CHOP. Meanwhile, D-M159 promoted the expression of IP3R1, GRP75, and VDAC1, which led to mitochondrial calcium iron overload, decreased mitochondrial membrane potential, and increased reactive oxygen species (ROS) generation, thereby activating the mitochondrial apoptotic pathway and inducing the aberrant expression of Bax, Bcl-2, Caspase-9, and Caspase-3. This study showed that D-M159 synergistically induced apoptosis in starved HeLa cells through endoplasmic reticulum stress and mitochondrial dysfunction, demonstrating its potential as a novel anticancer agent.

Exosomes are critical mediators of cell-to-cell communication in physiological and pathological processes, due to their ability to deliver a variety of bioactive molecules. Tumor-derived exosomes (TDEs), in particular, carry carcinogenic molecules that contribute to tumor progression, metastasis, immune escape, and drug resistance. Thus, TDE inhibition has emerged as a promising strategy to combat cancer. In this review, we discuss the key mechanisms of TDE biogenesis and secretion, emphasizing their implications in tumorigenesis and cancer progression. Moreover, we provide an overview of small-molecule TDE inhibitors that target specific biogenesis and/or secretion pathways, highlighting their potential use in cancer treatment. Lastly, we present the existing obstacles and propose corresponding remedies for the future development of TDE inhibitors.

To enhance cytoplasmic delivery efficiency, pH-sensitive liposomes (PSL) have been proposed as a novel strategy. To facilitate clinical translation, this study aims to understand the impact of both size and pH-sensitivity on cellular uptake pathways, intracellular trafficking and pharmacokinetics of liposomes. The large liposomes (130-160 nm) were prepared using thin-film hydration method, while small liposomes (∼60 nm) were fabricated using microfluidics, for both PSL and non-pH-sensitive liposomes (NPSL). Cellular uptake pathways and intracellular trafficking was investigated through confocal imaging with aid of various endocytosis inhibitors. Intracellular gemcitabine delivery by various liposomal formulations was quantified using HPLC, and the cytotoxicity was assessed via cell viability assays. Pharmacokinetics of gemcitabine loaded in various liposomes was evaluated in rats following intravenous administration. Larger liposomes had a higher loading capacity for hydrophilic gemcitabine (7% vs 4%). Small PSL exhibited superior cellular uptake compared to large PSL or NPSLs. Moreover, the alkalization of endosomes significantly attenuated the cellular uptake of PSL. Large liposomes (PSL and NPSL) predominantly entered cells via clathrin-dependent pathway, whereas small liposomes partially utilized caveolae-dependent pathway. However, the long circulation of the liposomes, as measured by the encapsulated gemcitabine, was compromised by both pH-sensitivity and size reduction (9.5 h vs 5.3 h). Despite this drawback, our results indicate that small PSL holds promise as vectors for the next generation of liposomal nanomedicine, owing to their superior cytoplasmic delivery efficiency.

Exosomal non-coding RNAs (ncRNAs), including miRNAs, lncRNAs, and circRNAs, have emerged as crucial modulators in cellular signaling, influencing wound healing processes. Stem cell-derived exosomes, which serve as vehicles for these ncRNAs, show remarkable therapeutic potential due to their ability to modulate wound healing stages, from initial inflammation to collagen formation. These ncRNAs act as molecular signals, regulating gene expression and protein synthesis necessary for cellular responses in healing. Wound healing is a complex, staged process involving inflammation, hemostasis, fibroblast proliferation, angiogenesis, and tissue remodeling. Stem cell-derived exosomal ncRNAs enhance these stages by reducing excessive inflammation, promoting anti-inflammatory responses, guiding fibroblast and keratinocyte maturation, enhancing vascularization, and ensuring organized collagen deposition. Their molecular cargo, particularly ncRNAs, specifically targets pathways to aid chronic wound repair and support scarless regeneration. This review delves into the unique composition and signaling roles of Stem cell-derived exosomes and ncRNAs, highlighting their impact across wound healing stages and their potential as innovative therapeutics. Understanding the interaction between exosomal ncRNAs and cellular signaling pathways opens new avenues in regenerative medicine, positioning Stem cell-derived exosomes and their ncRNAs as promising molecular-level interventions in wound healing.

Extracellular vesicles (EVs) contain bioactive lipids that play a key role in pathophysiology. We hypothesized that EVs released from salt-loaded hypertensive diabetic db/db mice have increased bioactive lipid content that inhibits intracellular calcium mobilization and increases the activity of renal epithelial sodium channels (ENaC). An enrichment of sphingomyelins (SMs) was found in small urinary EVs (uEVs) isolated from salt-loaded hypertensive diabetic db/db mice (n = 4) compared to non-salt loaded db/db mice with diabetes alone (n = 4). Both groups of mice were included in the same cohort to control for variability. Cultured mouse cortical collecting duct (mpkCCD) cells loaded with a calcium reporter dye and challenged with small uEVs from hypertensive diabetic db/db mice showed a decrease in calcium mobilization when compared to cells treated with small uEVs from diabetic db/db mice. The amiloride-sensitive transepithelial current was increased in mpkCCD cells treated with small uEVs with abundant sphingomyelin content from hypertensive diabetic db/db mice in a dose- and time-dependent manner. Similar results were observed in mpkCCD cells and Xenopus 2F3 cells treated with exogenous sphingomyelin in a time-dependent manner. Single-channel patch clamp studies showed a decrease in ENaC activity in cells transiently transfected with sphingomyelin synthase 1/2 specific siRNA compared to non-targeting siRNA. These data suggest EVs with high sphingomyelin content positively regulate renal ENaC activity in a mechanism involving an inhibition of calcium mobilization.

Extracellular vesicles (EVs) are heterogeneous vesicles released by donor cells that can be taken up by recipient cells, thus inducing cellular phenotype changes. Since their discovery decades ago, roles of EVs in modulating initiation, growth, survival and metastasis of cancer have been revealed. Recent studies from multifaceted perspectives have further detailed the contribution of EVs to cancer drug resistance; however, the role of EV uptake in conferring drug resistance seems to be overlooked. In this comprehensive review, we update the EV subtypes and approaches for determining EV uptake. The biological basis of EV uptake is systematically summarized. Moreover, we focus on the diverse uptake mechanisms by which EVs carry out the intracellular delivery of functional molecules and drug resistance signaling. Furthermore, we highlight how EV uptake confers drug resistance and identify potential strategies for targeting EV uptake to overcome drug resistance. Finally, we discuss the research gap on the role of EV uptake in promoting drug resistance. This updated knowledge provides a new avenue to overcome cancer drug resistance by targeting EV uptake.

Naturally occurring extracellular vesicles (EVs) and synthetic nanoparticles like liposomes have revolutionized precision diagnostics and medicine. EVs excel in biocompatibility and cell targeting, while liposomes offer enhanced drug loading capacity and scalability. The clinical translation of EVs is hindered by challenges including low yield and heterogeneity, whereas liposomes face rapid immune clearance and limited targeting efficiency. To bridge these gaps, biomimetic synthetic vesicles (SVs) have emerged as innovative platforms, combining the advantageous properties of EVs and liposomes. This review emphasizes critical aspects of EV biology, such as mechanisms of EV-cell interaction and source-dependent functionalities in targeting, immune modulation, and tissue regeneration, informing biomimetic SV engineering. We reviewed a broad array of biomimetic SVs, with a focus on lipid bilayered vesicles functionalized with proteins. These include cell-derived nanovesicles, protein-functionalized liposomes, and hybrid vesicles. By addressing current challenges and highlighting opportunities, this review aims to advance biomimetic SVs for transformative biomedical applications.

Extracellular vesicles may modify recipient cell behavior through multiple mechanisms, including interacting with the cell surface or internal membrane components and delivering luminal cargo to the cytoplasm. Here, we use a previously established ex vivo approach to investigate the cellular fate of EVs spiked into whole blood samples from nonhuman primate (NHP) and human donors and contrast these findings with results from in vitro assays. We report that EVs are internalized by NHP and human B cells while also associating to some degree with other PBMCs. EVs exhibit greater association with B cells in ex vivo whole blood compared to isolated B cells, suggesting that blood components may promote EV interactions or that cell isolation factors may inhibit this association. Cellular uptake of EVs involves clathrin-dependent endocytosis and may be aided by other pathways, including direct EV-cell membrane fusion. Overall, our data suggest that EV association, including uptake, by B cells occurs in at least two primate species. These findings highlight the potential to develop new strategies to either enhance or inhibit EV tropism toward B cells.

Exosomes, cell-derived vesicles produced by cells, are fascinating and drawing growing interest in biomedical exploration due to their exceptional properties. There is intriguing evidence that exosomes are involved in major biological processes, including diseases and regeneration. Exosomes from mesenchymal stem cells (MSCs) have shown promising outcomes in regenerative medicine. Numerous studies suggest that exosomes have several advantages over conventional synthetic nanocarriers, opening novel frontiers for innovative drug delivery. Regenerative medicine has demonstrated the profound therapeutic outcomes of engineered or loaded exosomes from MSCs. Different methods are being used to modify or/load exosomes. These exosomes can improve cell signaling pathways for bone and cartilage diseases, liver diseases, nerve tissues, kidney diseases, skin tissue, and cardiovascular diseases. Despite extensive research, clinical translation of these exosomes remains a challenge. The optimization of cargo loading methods, efficiency, physiological stability, and the isolation and characterization of exosomes present some challenges. The upcoming examination should include the development of large-scale, quality-controllable production approaches, the modification of drug loading approaches, and numerous in vivo investigations and clinical trials. Here, we provided an informative overview of the extracellular vesicles and modification/loading methods of exosomes. We discuss the last exosome research on regeneration disorders, highlighting the therapeutic applications of MSCs-derived exosomes. We also highlight future directions and challenges, underscoring the significance of addressing the main questions in the field.

Extracellular vesicles (EVs), nanoscale vesicles secreted by cells, have attracted considerable attention in recent years due to their role in tumor immunomodulation. These vesicles facilitate intercellular communication by transporting proteins, nucleic acids, and other biologically active substances, and they exhibit a dual role in tumor development and immune evasion mechanisms. Specifically, EVs can assist tumor cells in evading immune surveillance and attack by impairing immune cell function or modulating immunosuppressive pathways, thereby promoting tumor progression and metastasis. Conversely, they can also transport and release immunomodulatory factors that stimulate the activation and regulation of the immune system, enhancing the body's capacity to combat malignant diseases. This dual functionality of EVs presents promising avenues and targets for tumor immunotherapy. By examining the biological characteristics of EVs and their influence on tumor immunity, novel therapeutic strategies can be developed to improve the efficacy and relevance of cancer treatment. This review delineates the complex role of EVs in tumor immunomodulation and explores their potential implications for cancer therapeutic approaches, aiming to establish a theoretical foundation and provide practical insights for the advancement of future EVs-based cancer immunotherapy strategies.

Extracellular vesicles (EVs) are nanosized vesicles released by cells, containing molecular cargo such as proteins and nucleic acids. EVs offer promising avenues for the detection of biomarkers of disease and are excellent candidates for drug delivery and therapeutics. Although EVs can be obtained from biological fluids, it is challenging to obtain intact EVs from complex fluids and there is no universally accepted standard method of isolating EVs. When affinity chromatography-based isolation is used to isolate EVs from complex biofluids, there exist multiple ways to elute intact EVs from capture. This review aims to identify effective EV elution methods for preserving EV integrity and bioactivity after capture on aptamer-functionalized substrates, addressing the requirements of various downstream applications. We hypothesize that when used for elution, different materials and techniques influence the characteristics of EVs, such as their molecular content and bioactivity. The elution reagent and technique must be selected for the intended application for isolated EVs. However, currently, there is no agreement on the optimal elution method for EVs. This literature review aims to evaluate the different methods used to elute intact EVs from capture with regards to the downstream applications of isolated EVs. Based on the results of our analysis of recent literatures, the two elution reagents that are optimal for general purposes of the eluted intact EVs are deoxyribonuclease I and complementary oligonucleotides, as they both preserve EV characteristics that are required for molecular analysis and bioactivity, such as maintained morphology and protein profiles.

Photothermal therapy (PTT) is a method for treating cancer using the heat generated by light irradiation, often in combination with light-absorbing materials. Efficient PTT requires a drug delivery system to deliver light-absorbing materials to cancerous tissues. Gold nanostars (GNSs) enable efficient PTT through absorbing long-wavelength light. In this study, we established a platform for preparing GNSs-loaded extracellular vesicles (EVs), that were expected to provide the combined biological functionality of EVs and photothermal conversion properties. By electrostatically binding cationic polyethylene glycol (PEG)-modified GNSs (PEG-GNSs) to naturally negatively charged EVs, EV/PEG-GNSs nanocomplexes (EV-GNSs) were obtained by simply mixing the two components. The prepared EV-GNSs were approximately 200 nm in size and exhibited a negative surface charge. The surface protein marker of EVs, CD63, was detected using western blot, suggesting that the EVs were intact. In vitro evaluation showed that EV-GNSs exhibited better photothermal conversion properties than PEG-GNSs alone. When EV-GNSs were added to the cells, high laser-responsive cytotoxicity was observed following laser irradiation. Thus, EV-GNSs developed in this study may be suitable for PTT using gold-based nanoparticles.

Neutrophil membrane vesicles (NMVs) have been successfully applied to control the inflammatory cascade after spinal cord injury (SCI) by acting as an inflammatory factor decoy to front-load the overall inflammation regulatory window; however, the mechanisms by which NMVs regulate macrophage phenotypic shifts as well as their outcomes have rarely been reported. In this study, we demonstrated the "efferocytosis-like" effect of NMVs endocytosed by macrophages, supplementing the TCA cycle intermediate metabolite α-KG by promoting glutamine metabolism, which in turn facilitates oxidative phosphorylation and inhibits the NF-κB signaling pathway to reprogram inflammatory macrophages to the pro-regenerative phenotype. Based on these findings, a "Trojan horse" composite fiber scaffold was constructed; this comprised a carboxylated poly-l-lactic acid shell encapsulated with NMVs and a core loaded with brain-derived neurotrophic factor to spatiotemporally modulate the inflammatory microenvironment by 39.23% and sustainably promote nerve regeneration by 85.67%. In vivo experiments further confirmed the effect of NMV-coated fiber scaffolds on the regulation of early innate immune inflammation and the continuous promotion of nerve regeneration. This study not only further unravels the mechanism of neutrophil membrane-macrophage interactions but also provides a strategy for coordinating inflammatory reprogramming and nerve regeneration following SCI.

Ultrasound (US)-triggered cavitation of drug-loaded microbubbles (MBs) represents a promising approach for targeted drug delivery, with substantial benefits attainable through precise control over drug release dosage and form. This study investigates Camptothecin-loaded MBs (CPT-MBs) and Doxorubicin-loaded MBs (DOX-MBs), focusing on how properties such as hydrophilicity, hydrophobicity, and charged functional groups affect their interaction with the lipid surfaces of MBs, thereby influencing the fundamental characteristics and acoustic properties of the drug-loaded MBs. In comparison to DOX-MBs, CPT-MBs showed larger MB size (2.2 ± 0.3 and 1.4 ± 0.1 μm, respectively), a 2-fold increase in drug loading, and an 18 % reduction in leakage after 2 h at 37℃. Under 1 MHz US with a 100 ms pulse repetition interval (PRI), 1000 cycles, 5-minute duration, and 550 kPa acoustic pressure, CPT-MBs undergo inertial cavitation, while DOX-MBs undergo stable cavitation. Drug particles released from these MBs under US-induced cavitation were analyzed using dynamic light scattering, NanoSight, cryo-electron microscopy, and density gradient ultracentrifugation. Results showed that CPT-MBs mainly release free CPT, while DOX-MBs release multilayered DOX-lipid aggregates. The cytotoxicity to C6 cells induced by US-triggered cavitation of these two types of MBs also differed. DOX-lipid aggregates delayed initial uptake, leading to less pronounced short-term (2 h) effects compared to the rapid release of free CPT from CPT-MBs. These findings underscore the need to optimize drug delivery strategies by fine-tuning MB composition and US parameters to control drug release kinetics and achieve the best tumoricidal outcomes.

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.

Exosomes are small extracellular vesicles (EVs) constituting fully biological, cell-derived nanovesicles with great potential in cell-to-cell communication and drug delivery applications. The current gold standard for EV labeling and tracking is represented by fluorescent lipophilic dyes which, however, importantly lack selectivity, due to their unconditional affinity for lipids. Herein, an alternative EV fluorescent labeling approach is in-depth evaluated, by taking advantage of green fluorescent protein (GFP) farnesylation (GFP-f), a post-translational modification to directly anchor GFP to the EV membrane. The performance of GFP-f is analyzed, in terms of selectivity and efficiency, in several typical EV experimental setups such as delivery in recipient cells, surface engineering, and cargo loading. First, the capability of GFP and GFP-f to label exosomes was compared, showing significantly higher GFP protein levels and fluorescence intensity in GFP-f- than in GFP-labeled exosomes, highlighting the advantage of directly anchoring the GFP to the EV cell membrane. Then, the GFP-f tag was further compared to Vybrant DiD lipophilic dye labeling in exosome uptake studies, by capturing EV intracellular fluorescence in a time- and concentration-dependent manner. The internalization assay revealed a particular ability of GFP-f to monitor the uptake of tagged exosomes into recipient cells, with a significant peak of intensity reached 12 h after administration by GFP-f but not Vybrant-labeled EVs. Finally, the GFP-f labeling capability was challenged in the presence of a surface modification of exosomes and after transfection for siRNA loading. Results showed that both procedures can influence GFP-f performance compared to naïve GFP-f exosomes, although fluorescence is importantly maintained in both cases. Overall, these data provide direct insight into the advantages and limitations of GFP-f as a tagging protein for selectively and accurately tracking the exosome route from isolation to uptake in recipient cells, also in the context of EV bioengineering applications.

Growing micro- and nano-plastic (MNPs) pollution in the environment poses a threat to marine animals. Due to their excellent filtration capacity, bivalves can easily ingest MNPs, which could be translocated to open circulation system with potential risks. In the present study, the accumulation and elimination of MNPs (200 nm and 1 µm) in the mussel hemolymph serum and hemocytes were firstly quantified, and the differential sensitiveresponses of two subpopulations of hemocytes were then explored by in vivo exposure under environmentally relevant concentration of MNPs (200 µg/L). We demonstrated that MNPs were readily translocated into hemolymph serum, but were immediately followed by efficient internalization by hemocytes. Remarkably, concentrations of MNPs in hemolymph were only 0.63 and 0.39 times lower than the ambient exposure concentration. Granulocytes displayed a much higher potential of accumulating MNPs than the agranulocytes. MPs were more readily internalized by granulocytes, with their estimated maximum bioaccumulation factor (BCF) of 0.29 L/g. Due to the primary function of phagocytic encapsulation of MNPs by granulocytes, lysosome features especially the decline of subsequent lysosome membrane potential could be a potential sensitive biomarker in response to MNPs exposure. Our results provided insights on the bioaccumulation of MNPs at the cellular levels in marine bivalves.

The study of extracellular vesicles has become an incredibly important field of study, but the inherent heterogeneity of these vesicles continues to make their study challenging. The genetic variability and well-documented protocols for the growth and vesicle isolation from Leishmania parasites provide a unique opportunity to compare the heterogeneity of different populations secreted by Leishmania clones. Leishmania mexicana was cultured on solid SDM agar plates and 8 clonal colonies were selected. The EVs collected from the liquid cultures of these 8 clones were assessed by NTA, TEM, and proteomic analysis. We found that all 8 clonal L. mexicana cultures were visually indistinguishable from each other and had similar growth rate, and these physical similarities extended to their EVs. However, proteomic analysis reveals that the EVs collected have unique protein profiles compared to each other and EVs isolated from a heterogeneous liquid culture of L. mexicana. We selected 3 clonal EVs for further mouse infection experiments and found that EVs from CL7 L. mexicana consistently caused reduced footpad swelling in C57BL6 mice footpads compared to EVs from CL1, CL8, and heterogenous L. mexicana. This trend was not observed when infecting Balb/C mice and C57BL6 with the parasites alone, with only CL1 L. mexicana causing significantly increased infection in Balb/c mice. Our results together show that EVs isolated from different clonal colonies of L. mexicana have distinct differences in protein cargo which can lead to varying outcomes on Leishmania infection. Further evaluation will be needed to determine the underlying mechanisms behind this and verify that differences observed in infectivity are directly caused by variations between our L. mexicana clones, especially genetic sequencing and immunoblotting to validate our results.

Mesenchymal stem cells (MSCs) are highly valued in regenerative medicine due to their ability to self-renew and differentiate into various cell types. Their therapeutic benefits are primarily due to their paracrine effects, in particular through extracellular vesicles (EVs), which are related to intercellular communication. Recent advances in EV production and extraction technologies highlight the potential of MSC-derived EVs (MSC-EVs) in tissue engineering and regenerative medicine. MSC-EVs offer several advantages over traditional cell therapies, including reduced toxicity and immunogenicity compared with whole MSCs. EVs carrying functional molecules such as growth factors, cytokines, and miRNAs play beneficial roles in tissue repair, fibrosis treatment, and scar prevention by promoting angiogenesis, skin cell migration, proliferation, extracellular matrix remodeling, and reducing inflammation. Despite the potential of MSC-EVs, there are several limitations to their use, including variability in quality, the need for standardized methods, low yield, and concerns about the composition of EVs and the potential risks. Overall, MSC-EVs are a promising alternative to cell-based therapies, and ongoing studies aim to understand their actions and optimize their use for better clinical outcomes in wound healing and skin regeneration.

Extracellular vesicles (EVs) are increasingly investigated for delivering nucleic acid (NA) therapeutics, leveraging their natural role in transporting NA and protein-based cargo in cell-to-cell signaling. Their synthetic counterparts, lipid nanoparticles (LNPs), have been developed over the past decades as NA carriers, culminating in the approval of several marketed formulations such as patisiran/Onpattro® and the mRNA-1273/BNT162 COVID-19 vaccines. The success of LNPs has sparked efforts to develop innovative technologies to target extrahepatic organs, and to deliver novel therapeutic modalities, such as tools for in vivo gene editing. Fueled by the recent advancements in both fields, this review aims to provide a comprehensive overview of the basic characteristics of EV and LNP-based NA delivery systems, from EV biogenesis to structural properties of LNPs. It addresses the primary challenges encountered in utilizing these nanocarriers from a drug formulation and delivery perspective. Additionally, biodistribution profiles, in vitro and in vivo transfection outcomes, as well as their status in clinical trials are compared. Overall, this review provides insights into promising research avenues and potential dead ends for EV and LNP-based NA delivery systems.

Extracellular vesicles (EVs) are involved in cell-to-cell communication. Following uptake, EV cargo molecules, including DNA, RNA, lipids, and proteins, influence gene expression and molecular signaling in recipient cells. Although various studies have identified disease-specific EV molecules, further research into their biogenesis and secretion mechanisms is needed for clinical application. Here, we investigated the role of UPF2 in regulating the biogenesis and components of EVs. Notably, UPF2 promoted the expression of CD81, a membrane protein marker of EVs, as UPF2 silencing decreased CD81 levels in EVs, both inside the cell and secreted. In contrast, the expression levels of CD63 increased, without altering the size or numbers of EVs. In addition, reducing UPF2 levels did not affect the total number of EVs but lowered production of CD81-positive EVs and reduced the efficiency of uptake by recipient cells. Collectively, our findings uncover a novel function for UPF2 in regulating the production of CD81 and changing EV properties.

Triglyceride-based lipid emulsions are critical for total parenteral nutrition (TPN), but their long-term use has adverse effects, such as severe liver dysfunction necessitating improved formulations. This study compares the uptake mechanism and intracellular fate of novel glycerol-stabilized nano-sized lipid emulsions with conventional emulsions in CD4+ T cells, focusing on their impact on cellular metabolism.

Nanoemulsions were formulated with increased glycerol content. Uptake of emulsions in primary human CD4+ T cells was investigated using different endocytic blockers, then quantified by flow cytometry, and visualized by confocal microscopy. To investigate emulsion intracellular fate, fatty acids in membrane phospholipids were quantified by GC-MS/MS and cellular metabolism was assessed by Seahorse technology. Results show T cells internalize both conventional and nano-sized emulsions using macropinocytosis. Fatty acids from emulsions are stored as neutral lipids in intracellular vesicles and are incorporated into phospholipids of cellular membranes. However, only nanoemulsions additionally use clathrin-mediated endocytosis and deliver fatty acids to mitochondria for increased β-oxidation.

Size of lipid emulsion droplets significantly influences their uptake and subsequent metabolism in CD4+ T cells. Our results highlight the potential for improved nutrient utilization with nanoemulsions in TPN formulations possibly leading to less adverse effects.

Extracellular vesicles (EVs) are a population of vesicular bodies originating from cells, and EVs have been proven to have the potential to deliver different cargos, such as RNAs. However, conventional methods are not able to encapsulate long RNAs into EVs efficiently or may compromise the integrity of EVs. In this study, we have devised a strategy to encapsulate long circRNAs (>1000 nt) into EVs by harnessing the sorting mechanisms of cells. This strategy utilizes the inherent richness of circular RNAs in EVs and a genetic engineering method to increase the cytoplasmic concentration of target circRNAs, facilitating highly efficient RNA back-splicing to drive the circularization of RNAs. This allows target circRNAs to load into EVs with high efficiency. Furthermore, we demonstrate the practical applications of this strategy, showing that these circRNAs can be delivered by EVs to recipient cells for protein expression and to mice for gene editing.

Peptide-based drugs hold great potential for cancer treatment, and their effectiveness is driven by mechanisms on how peptides target cancer cells and escape from potential lysosomal entrapment post-endocytosis. Yet, the mechanisms remain elusive, which hinder the design of peptide-based drugs. Here hendeca-arginine peptides (R11) are synthesized for targeted delivery in bladder carcinoma (BC), investigated the targeting efficiency and elucidated the mechanism of peptide-based delivery, with the aim of refining the design and efficacy of peptide-based therapeutics. It is demonstrated that the over-activated Piezo1/integrin β1 (ITGB1) signaling axis significantly facilitates tumor-targeted delivery of R11 peptides via macropinocytosis. Furthermore, R11 peptides formed hydrogen bonds with integrin β1, facilitating targeting and penetration into tumor cells. Additionally, R11 peptides protected integrin β1 from lysosome degradation, promoting its recycling from cytoplasm to membrane. Moreover, this findings establish a positive feedback loop wherein R11 peptides activate Piezo1 by increasing membrane fusion, promoting Ca2+ releasing and resulting in enhanced integrin β1-mediated endocytosis in both orthotopic models and clinical tissues, demonstrating effective tumor-targeted delivery. Eventually, the Piezo1/integrin β1 signaling axis promoted cellular uptake and transport of peptides, establishing a positive feedback loop, promoting mechanical delivery to cancer and offering possibilities for drug modification in cancer therapy.

Nanotechnology has brought about significant progress through the use of goods based on nanomaterials. However, concerns remain about the accumulation of these materials in the environment and their potential toxicity to living organisms. Plants have the ability to take in nanomaterials (NMs), which can cause changes in their physiology and morphology. On the other hand, nanoparticles (NPs) have been used to increase plant development and control pests in agriculture by including them into agrochemicals. The challenges of the interaction, internalization, and accumulation of NMs within plant tissues are enormous, mainly because of the various characteristics of NMs and the absence of reliable analytical tools. As our knowledge of the interactions between NMs and plant cells expands, we are able to create novel NMs that are tailored, targeted, and designed to be safe, thus minimizing the environmental consequences of nanomaterials. This review provides a thorough examination and comparison of the main microscopy techniques, spectroscopic methods, and far-field super-resolution methodologies used to examine nanomaterials within the cell walls of plants.

During cell invasion, large Extracellular Vesicle (lEV) release from host cells was dose-dependently triggered by Trypanosoma cruzi metacyclic trypomastigotes (Mtr). This lEV release was inhibited when IP3-mediated Ca2+ exit from the ER and further Ca2+ entry from plasma membrane channels was blocked, but whilst any store-independent Ca2+ entry (SICE) could continue unabated. That lEV release was equally inhibited if all entry from external sources was blocked by chelation of external Ca2+ points to the major contributor to Mtr-triggered host cell lEV release being IP3/store-mediated Ca2+ release, SICE playing a minor role. Host cell lEVs were released through Mtr interaction with host cell lipid raft domains, integrins, and mechanosensitive ion channels, whereupon [Ca2+]cyt increased (50 to 750 nM) within 15 s. lEV release and cell entry of T. cruzi, which increased up to 30 and 60 mpi, respectively, as well as raised actin depolymerization at 60 mpi, were all reduced by TRPC inhibitor, GsMTx-4. Vesicle release and infection was also reduced with RGD peptide, methyl-β-cyclodextrin, knockdown of calpain and with the calpain inhibitor, calpeptin. Restoration of lEV levels, whether with lEVs from infected or uninfected epithelial cells, did not restore invasion, but supplementation with lEVs from infected monocytes, did. We provide evidence of THP-1 monocyte-derived lEV interaction with Mtr (lipid mixing by R18-dequenching; flow cytometry showing transfer to Mtr of R18 from R18-lEVs and of LAP(TGF-β1). Active, mature TGF-β1 (at 175 pg/×105 in THP-1 lEVs) was detected in concentrated lEV-/cell-free supernatant by western blotting, only after THP-1 lEVs had interacted with Mtr. The TGF-β1 receptor (TβRI) inhibitor, SB-431542, reduced the enhanced cellular invasion due to monocyte-lEVs.

The translation of pre-clinical anti-cancer therapies to regulatory approval has been promising, but slower than hoped. While innovative and effective treatments continue to achieve or seek approval, setbacks are often attributed to a lack of efficacy, failure to achieve clinical endpoints, and dose-limiting toxicities. Successful efforts have been characterized by the development of therapeutics designed to specifically deliver optimal and effective dosing to tumour cells while minimizing off-target toxicity. Much effort has been devoted to the rational design and application of synthetic nanoparticles to serve as targeted therapeutic delivery vehicles. Several challenges to the successful application of this modality as delivery vehicles include the induction of a protracted immune response that results in their rapid systemic clearance, manufacturing cost, lack of stability, and their biocompatibility. Extracellular vesicles (EVs) are a heterogeneous class of endogenous biologically produced lipid bilayer nanoparticles that mediate intercellular communication by carrying bioactive macromolecules capable of modifying cellular phenotypes to local and distant cells. By genetic, chemical, or metabolic methods, extracellular vesicles (EVs) can be engineered to display targeting moieties on their surface while transporting specific cargo to modulate pathological processes following uptake by target cell populations. This review will survey the types of EVs, their composition and cargoes, strategies employed to increase their targeting, uptake, and cargo release, and their potential as targeted anti-cancer therapeutic delivery vehicles.

Extracellular vesicles (EVs) are an emerging class of drug carriers and are primarily reported to be internalized into recipient cells via a combination of endocytic routes such as clathrin-mediated, caveolae-mediated and macropinocytosis pathways. In this work, (1) we investigated potential effects of homotypic vs. heterotypic interactions by studying the cellular uptake of homologous EVs (EV donor cells and recipient cells of the same type) vs. heterologous EVs (EV donor cells and recipient cells of different types) and (2) determined the route of EV internalization into low pinocytic/hard-to-deliver cell models such as brain endothelial cells (BECs). Homotypic interactions led to a greater extent of uptake into the recipient BECs compared to heterotypic interactions. However, we did not see a complete reduction in EV uptake into recipient BECs when endocytic pathways were blocked using pharmacological inhibitors and our findings from a R18-based fusion assay suggest that EVs primarily use membrane fusion to enter low-pinocytic recipient BECs instead of relying on endocytosis. Lipophilic PKH67 dye-labeled EVs but not intravesicular esterase-activated calcein ester-labeled EVs severely reduced particle uptake into BECs while phagocytic macrophages internalized EVs labeled with both dyes to comparable extents. Our results also highlight the importance of carefully choosing labeling dye chemistry to study EV uptake, especially in the case of low pinocytic cells such as BECs.

As a new mechanism of intercellular communication, the uptake of extracellular vesicles (EVs) by receptor cells has become a hot topic in the field. Previously, research on the uptake of EVs has focused on the mechanism of small EVs (sEVs, also known as exosomes). As sEVs represent a mixed heterogeneous population, the issue of whether there are different uptake mechanisms for different subsets of sEVs by recipient cells urgently need to be addressed. There are EVs in follicular fluid, which play an important role in the communication between follicular cells and the development of oocytes. Previously, we isolated two subtypes of sEVs in follicular fluid: low density-sEVs (LD-sEVs) and high density-sEVs (HD-sEVs). The current study aimed to explore the uptake characteristics of these two subtypes of sEVs by granulosa cells. First, PKH67 was used to label the two sEVs subtypes, and we observed their uptake by granulosa cells using confocal microscopy and flow cytometry. We then explored the specific mechanisms underlying uptake of these two sEV subtypes by granulosa cells using specific inhibitors and RNA interference. The results showed that granulosa cells took up both kinds of sEVs through a clathrin-independent pathway. In addition to requiring caveolin, cholesterol, and Na+/H+ exchange, the uptake of HD-sEVs also depended on the activity of tyrosine kinase and phosphoinositide 3-kinase. A better understanding of the mechanism of granulosa cell uptake of different subtypes of sEVs in follicular fluid is of considerable significance leading to more accurate use of EVs for targeted treatment of infertility and other related diseases.

The study of host-pathogen interactions has increased considerably in recent decades. This intercellular communication has been mediated by extracellular vesicles (EVs) that play an important role during the interaction. EVs are particles of lipid bilayer and described in different types of cells, eukaryotic or prokaryotic. Depending on their biogenesis they are described as exosomes (derived from multivesicular bodies) and microvesicles (derived from the plasma membrane). The EVs carry biomolecules, including nucleic acids, lipids, and proteins that can be released or internalized by other cells in different pathways (endocytosis, macropinocytosis, phagocytosis, or membrane fusion) in the process described as uptake. The balance between biogenesis and uptake of EVs could modify physiological and pathophysiological processes of the cell. This review is focusing on the dynamic roles of release and capture of EVs during host-pathogen interaction. We also do a critical analysis of methodologies for obtaining and analyzing EVs. Finally, we draw attention to critical points to be considered in EV biogenesis and uptake studies.