Apoptotic body-inspired nanotherapeutics efficiently attenuate osteoarthritis by targeting BRD4-regulated synovial macrophage polarization.

Bromodomain-containing protein 4 (BRD4) is the most well-studied BET protein that is important for the innate immune response. We recently revealed that targeting BRD4 triggers apoptosis in tumor-associated macrophages, but its role in synovial macrophages and joint inflammation is largely unknown. Herein, we demonstrated that BRD4 was highly expressed in the iNOS-positive M1 macrophages in the human and mouse osteoarthritis (OA) synovium, and conditional knockout of BRD4 in the myeloid lineage using Lyz2-cre; BRD4flox/flox mice significantly abolished anterior cruciate ligament transection (ACLT)-induced M1 macrophage accumulation and synovial inflammation. Accordingly, we successfully constructed apoptotic body-inspired phosphatidylserine-containing nanoliposomes (PSLs) loaded with the BRD4 inhibitor JQ1 to regulate inflammatory macrophages. JQ1-loaded PSLs (JQ1@PSLs) exhibited a higher cellular uptake by macrophages than fibroblast-like synoviocytes (FLSs) in vitro and in vivo, as well as the reduction in proinflammatory M1 macrophage polarization. Intra-articular injections of JQ1@PSLs showed prolonged retention within the joint, and remarkably reduced synovial inflammation and joint pain via suppressing M1 polarization accompanied by reduced TRPA1 expression by targeted inhibition of BRD4 in the macrophages, thus attenuating cartilage degradation during OA development. The results show that BRD4-inhibiting JQ1@PSLs can targeted-modulate macrophage polarization, which opens a new avenue for efficient OA therapy via a "Trojan horse".

Immunomodulatory function of licensed human bone marrow mesenchymal stromal cell-derived apoptotic bodies.

BACKGROUND: Mesenchymal stromal cells (MSCs) show great potential for immunomodulatory and anti-inflammatory treatments. Clinical trials have been performed for the treatment of Type 1 diabetes, graft-versus-host disease and organ transplantation, which offer a promise of MSCs as an immunomodulatory therapy. Nevertheless, their unstable efficacy and immunogenicity concerns present challenges to clinical translation. It has emerged that the MSC-derived secretome, which includes secreted proteins, exosomes, apoptotic bodies (ABs) and other macromolecules, may have similar therapeutic effects to parent MSCs. Among all of the components of the MSC-derived secretome, most interest thus far has been garnered by exosomes for their therapeutic potential. However, since MSCs were reported to undergo apoptosis after in vivo transplantation and release ABs, we speculated as to whether ABs have immunomodulatory effects. In this study, cytokine licensing was used to enhance the immunomodulatory potency of MSCs and ABs derived from licensed MSCs in vitro were isolated to explore their immunomodulatory effects as an effective non-viable cell therapy. RESULTS: IFN-γ and IFN-γ/TGF-ß1 licensing enhanced the immunomodulatory effect of MSCs on T cell proliferation. Further, TGF-ß1 and IFN-γ licensing strengthened the immunomodulatory effect of MSC on reducing the TNF-α and IL-1ß expression by M1 macrophage-like THP-1 cells. Additionally, we discovered the immunomodulatory effect mediated by MSC-derived apoptotic bodies. Licensing impacted the uptake of ABs by recipient immune cells and importantly altered their phenotypes. CONCLUSION: ABs derived from IFN-γ/TGF-ß1-licensed apoptotic MSCs significantly inhibited T cell proliferation, induced more regulatory T cells, and maintained immunomodulatory T cells but reduced pro-inflammatory T cells.

Apoptotic bodies of bone marrow mesenchymal stem cells inhibit endometrial stromal cell fibrosis by mediating the Wnt/ß-catenin signaling pathway.

Background: Intrauterine adhesions (IUAs) are a common illness of the uterine cavity. Endometrial fibrosis is the main pathological feature. In addition to a high recurrence rate, patients with severe IUAs have a low pregnancy rate. However, there are few effective treatments for IUAs. This study aims to confirm the influence of apoptotic bodies of bone marrow mesenchymal stem cells (BMSCs) on endometrial stromal cell fibrosis by mediating the Wnt/ß-catenin signaling pathway and to provide new insight for the clinical treatment of IUAs. Methods: Human endometrial stromal cells (HESCs) were used to establish an IUA cell model by treatment with TGF-ß1, and a rat IUA model was established by the double injury method. Apoptosis of BMSCs was detected by TUNEL assays, and cell morphology was observed by the CM-DiI tracer. The morphology of apoptotic vacuoles and apoptotic bodies (ABs) was detected by TEM. We used Western blotting to detect the expression of histone H3.3, histone H2B, C3b, cyclin D1, C1QC, α-SMA, COL1A1, COL5A2, FN, CTGF, Wnt2b, c-MYC, CK-18 and VIM. The expression levels of α-SMA, COL1A1, COL5A2, FN and CTGF were detected by RT‒qPCR. The expression levels of α-SMA, COL1A1, FN and CTGF were detected by immunofluorescence. Immunohistochemistry was used to detect the expression of TGF-ß, CK-18 and VIM. Flow cytometry, cell scratch assays, CCK-8 assays, and H & E and Masson staining were used to detect the cell cycle, cell migration, cell proliferation, and endometrial pathology, respectively. Results: We found that ultraviolet light (UV) irradiation induced apoptosis of BMSCs and increased the production of ABs. TGF-ß1 treatment can induce HESCs to form extracellular matrix (ECM), and aggravate cell fibrosis, and adding ABs or FH535, an inhibitor of the Wnt/ß-catenin signaling pathway, can inhibit TGF-ß1-induced HESC fibrosis. However, the inhibitory effect of ABs on TGF-ß1-induced fibrosis of HESCs was attenuated by the addition of LiCl. In the Wnt/ß-catenin signaling pathway, LiCl is an activator after coculture with TGF-ß1. In vivo, IUA-induced narrowing of the uterine cavity was accompanied by intrauterine adhesions, increased deposition of collagen fibers, upregulation of TGF-ß1, VIM, α-SMA, COL1A1 and COL5A2, and downregulation of CK-18. These changes in expression were reversed after treatment with ABs or FH535. When ABs and LiCl were added at the same time, the inhibitory effect of ABs on IUA fibrosis was weakened. Conclusion: BMSC-derived ABs inhibit the fibrosis of HESCs by inhibiting the Wnt/ß-catenin signaling pathway. These results provide a new direction for the clinical treatment of IUAs.

Osteoclast-derived apoptotic bodies inhibit naive CD8+ T cell activation via Siglec15, promoting breast cancer secondary metastasis.

The bone microenvironment promotes cancer cell proliferation and dissemination. During periodic bone remodeling, osteoclasts undergo apoptosis, producing large numbers of apoptotic bodies (ABs). However, the biological role of osteoclast-derived ABs, which are residents of the bone-tumor niche, remains largely unknown. Here, we discover that AB-null MRL/lpr mice show resistance to breast cancer cell implantation, with more CD8+ T cell infiltrations and a higher survival rate. We uncover that the membranous Siglec15 on osteoclast-derived ABs binds with sialylated Toll-like receptor 2 (TLR2) and blocks downstream co-stimulatory signaling, leading to the inhibition of naive CD8+ T cell activation. In addition, our study shows that treatment with Siglec15 neutralizing antibodies significantly reduces the incidence of secondary metastases and improves the survival rate of mice with advanced breast cancer bone metastasis. Our findings reveal the immunosuppressive function of osteoclast-derived ABs in the bone-tumor niche and demonstrate the potential of Siglec15 as a common target for anti-resorption and immunotherapy.

The miR-21-5p enriched in the apoptotic bodies of M2 macrophage-derived extracellular vesicles alleviates osteoarthritis by changing macrophage phenotype.

Macrophages (Mφs) play a crucial role in the pathological progression of osteoarthritis (OA) by regulating inflammation and tissue repair. Decreasing pro-inflammatory M1-Mφs and increasing anti-inflammatory M2-Mφs can alleviate OA-related inflammation and promote cartilage repair. Apoptosis is a natural process associated with tissue repair. A large number of apoptotic bodies (ABs), a type of extracellular vesicle, are produced during apoptosis, and this is associated with a reduction in inflammation. However, the functions of apoptotic bodies remain largely unknown. In this study, we investigated the role of M2-Mφs-derived apoptotic bodies (M2-ABs) in regulating the M1/M2 balance of macrophages in a mouse model of OA. Our data show that M2-ABs can be targeted for uptake by M1-Mφs, and this reprograms M1-to-M2 phenotypes within 24 h. The M2-ABs significantly ameliorated the severity of OA, alleviated the M1-mediated pro-inflammatory environment, and inhibited chondrocyte apoptosis in mice. RNA-seq revealed that M2-ABs were enriched with miR-21-5p, a microRNA that is negatively correlated with articular cartilage degeneration. Inhibiting the function of miR-21-5p in M1-Mφs significantly reduced M2-ABs-guided M1-to-M2 reprogramming following in vitro cell transfection. Together, these results suggest that M2-derived apoptotic bodies can prevent articular cartilage damage and improve gait abnormalities in OA mice by reversing the inflammatory response caused by M1 macrophages. The mechanism underlying these findings may be related to miR-21-5p-regulated inhibition of inflammatory factors. The application of M2-ABs may represent a novel cell therapy, and could provide a valuable strategy for the treatment of OA and/or chronic inflammation.

Apoptotic extracellular vesicle formation via local phosphatidylserine exposure drives efficient cell extrusion.

Cell extrusion is a universal mode of cell removal from tissues, and it plays an important role in regulating cell numbers and eliminating unwanted cells. However, the underlying mechanisms of cell delamination from the cell layer are unclear. Here, we report a conserved execution mechanism of apoptotic cell extrusion. We found extracellular vesicle (EV) formation in extruding mammalian and Drosophila cells at a site opposite to the extrusion direction. Lipid-scramblase-mediated local exposure of phosphatidylserine is responsible for EV formation and is crucial for executing cell extrusion. Inhibition of this process disrupts prompt cell delamination and tissue homeostasis. Although the EV has hallmarks of an apoptotic body, its formation is governed by the mechanism of microvesicle formation. Experimental and mathematical modeling analysis illustrated that EV formation promotes neighboring cells' invasion. This study showed that membrane dynamics play a crucial role in cell exit by connecting the actions of the extruding cell and neighboring cells.

Ce6/PTX2-NP/G@NHs Confer Radiosensitivity in Non-Small Cell Lung Cancer via Promotion of Apoptotic Body-Mediated Neighboring Effects.

This study fabricates a nanoparticle delivery system of gold nanoparticles-dextran nanoparticles loaded with hypoxia-activated paclitaxel dimeric prodrug nanoparticles (PTX2-NP) and photosensitizer chlorin e6/paclitaxel-nanoparticle/gold@N-(2-hydroxypropyl) (Ce6/PTX2-NP/G@NHs) and analyzed the possible molecular mechanism for enhancing the radiosensitivity of non-small cell lung cancer (NSCLC). Ce6/PTX2-NP/G@NHs were prepared by a coupling reaction and dextran inclusion, followed by characterization using spectroscopy techniques. The cellular uptake and cytotoxicity of Ce6/PTX2-NP/G@NHs were analyzed. Radiosensitizing effects of the nanoparticles were evaluated by determining the malignant phenotypes and reactive oxygen species production of A549 cells and PI3K/AKT pathway-related proteins under 685 nm laser irradiation. A549 tumor-bearing nude mice were modeled to further confirm the radiosensitizing effect. Ce6/PTX2-NP/G@NHs were effectively internalized by A549 cells, producing cytotoxicity under laser irradiation. Ce6/PTX2-NP/G@NHs reduced cell viability, clonogenic potential, migration, and invasion along with reactive oxygen species (ROS) production while promoting apoptosis in A549 cells under laser irradiation. By inhibiting the PI3K/AKT pathway, Ce6/PTX2-NP/G@NHs increased the sensitivity of A549 cells to radiotherapy where apoptotic body (ApoBD)-mediated neighboring effects also played a key role. Ce6/PTX2-NP/G@NHs accumulated in tumor sites of nude mice and enhanced the radiosensitivity of NSCLC. Ce6/PTX2-NP/G@NHs showed no obvious toxicity or side effects in vivo. Collectively, the new Ce6/PTX2-NP/G@NHs nanoparticle delivery system can enhance the radiosensitivity of NSCLC via the promotion of ApoBD-mediated neighboring effects and inactivation of the PI3K/AKT pathway.

"Find-eat" strategy targeting endothelial cells via receptor functionalized apoptotic body nanovesicle.

Endothelial cell (EC) injury plays a key role in the chronic wound process. A long-term hypoxic microenvironment hinders the vascularization of ECs, thus delaying wound healing. In this study, CX3CL1-functionalized apoptotic body nanovesicles (nABs) were constructed. The "Find-eat" strategy was implemented through a receptor-ligand combination to target ECs that highly express CX3CR1 in the hypoxic microenvironment, therefore amplifying the "Find-eat" signal and promoting angiogenesis. Apoptotic bodies (ABs) were obtained by chemically inducing apoptosis of adipose-derived stem cells (ADSCs), and then functionalized nABs containing deferoxamine (DFO-nABs) were obtained through a series of steps, including optimized hypotonic treatment, mild ultrasound, drug mixing and extrusion treatment. In vitro experiments showed that nABs had good biocompatibility and an effective "Find-eat" signal via CX3CL1/CX3CR1 to induce ECs in the hypoxic microenvironment, thereby promoting cell proliferation, cell migration, and tube formation. In vivo experiments showed that nABs could promote the rapid closure of wounds, release the "Find-eat" signal to target ECs and realize the sustained release of angiogenic drugs to promote new blood vessel formation in diabetic wounds. These receptor-functionalized nABs, which can target ECs by releasing dual signals and achieve the sustained release of angiogenic drugs, may provide a novel strategy for chronic diabetic wound healing.

Apoptotic Body-Rich Media from Tenocytes Enhance Proliferation and Migration of Tenocytes and Bone Marrow Stromal Cells.

The intrinsic healing following tendon injury is ideal, in which tendon progenitor cells proliferate and migrate to the injury site to directly bridge or regenerate tendon tissue. However, the mechanism determining why and how those cells are attracted to the injury site for tendon healing is not understood. Since the tenocytes near the injury site go through apoptosis or necrosis following injury, we hypothesized that secretions from injured tenocytes might have biological effects on cell proliferation and migration to enhance tendon healing. Tenocyte apoptosis was induced by 24 h cell starvation. Apoptotic body-rich media (T-ABRM) and apoptotic body-depleted media (T-ABDM) were collected from culture media after centrifuging. Tenocytes and bone marrow-derived stem cells (BMDSCs) were isolated and cultured with the following four media: (1) T-ABRM, (2) T-ABDM, (3) GDF-5, or (4) basal medium with 2% fetal calf serum (FCS). The cell activities and functions were evaluated. Both T-ABRM and T-ABDM treatments significantly stimulated the cell proliferation, migration, and extracellular matrix synthesis for both tenocytes and BMDSCs compared to the control groups (GDF-5 and basal medium). However, cell proliferation, migration, and extracellular matrix production of T-ABRM-treated cells were significantly higher than the T-ABDM, which indicates the apoptotic bodies are critical for cell activities. Our study revealed the possible mechanism of the intrinsic healing of the tendon in which apoptotic bodies, in the process of apoptosis, following tendon injury promote tenocyte and stromal cell proliferation, migration, and production. Future studies should analyze the components of the apoptotic bodies that play this role, and, thus, the targeting of therapeutics can be developed.

Resistance exercise differentially alters extracellular vesicle size and subpopulation characteristics in healthy men and women: an observational cohort study.

Extracellular vesicles (EVs) are established mediators of adaptation to exercise. Currently, there are no published data comparing changes in EVs between men and women after resistance exercise. We tested the hypothesis that EV profiles would demonstrate a sex-specific signature following resistance exercise. Ten men and 10 women completed an acute heavy resistance exercise test for back squats using 75% of their one-repetition maximum. Blood was drawn before and immediately after exercise. EVs were isolated from plasma using size exclusion chromatography and stained with antibodies associated with exosomes (CD63), microvesicles (VAMP3), apoptotic bodies (THSD1), and a marker for skeletal muscle EVs (SGCA). CD63+ EV concentration and proportion of total EVs increased 23% (P = 0.006) and 113% (P = 0.005) in both sexes. EV mean size declined in men (P = 0.020), but not in women, suggesting a relative increase in small EVs in men. VAMP3+ EV concentration and proportion of total EVs increased by 93% (P = 0.025) and 61% (P = 0.030) in men and women, respectively. SGCA+ EV concentration was 69% higher in women compared with men independent of time (P = 0.007). Differences were also observed for CD63, VAMP3, and SGCA median fluorescence intensity, suggesting altered surface protein density according to sex and time. There were no significant effects of time or sex on THSD1+ EVs or fluorescence intensity. EV profiles, particularly among exosome-associated and muscle-derived EVs, exhibit sex-specific differences in response to resistance exercise which should be further studied to understand their relationship to training adaptations.

The expanding family of neutrophil-derived extracellular vesicles.

Neutrophils are immune cells involved in several inflammatory and homeostatic processes. Their capacity to release cargo can be classified based on whether the cargo is released on its own, or in conjunction with plasma membrane structures. Examples of plasma membrane-free secretion modes are degranulation, neutrophil extracellular trap (NET) release, and cytokine release through inflammasome formation. The most studied membrane-covered neutrophil-derived structures are exosomes and ectosomes that are collectively called extracellular vesicles (EV). Apoptotic vesicles are another recognized EV subtype. Over the last decade, additional membrane-covered neutrophil-derived structures were characterized: migratory cytoplasts, migrasomes, and elongated neutrophil-derived structures (ENDS). All these structures are smaller than the neutrophils, cannot reproduce themselves, and thus meet the latest consensus definition of EVs. In this review, we focus on the less well-studied neutrophil EVs: apoptotic vesicles, cytoplasts, migrasomes, and ENDS.

Bracovirus Sneaks Into Apoptotic Bodies Transmitting Immunosuppressive Signaling Driven by Integration-Mediated eIF5A Hypusination.

A typical characteristics of polydnavirus (PDV) infection is a persistent immunosuppression, governed by the viral integration and expression of virulence genes. Recently, activation of caspase-3 by Microplitis bicoloratus bracovirus (MbBV) to cleave Innexins, gap junction proteins, has been highlighted, further promoting apoptotic cell disassembly and apoptotic body (AB) formation. However, whether ABs play a role in immune suppression remains to be determined. Herein, we show that ABs transmitted immunosuppressive signaling, causing recipient cells to undergo apoptosis and dismigration. Furthermore, the insertion of viral-host integrated motif sites damaged the host genome, stimulating eIF5A nucleocytoplasmic transport and activating the eIF5A-hypusination translation pathway. This pathway specifically translates apoptosis-related host proteins, such as P53, CypA, CypD, and CypJ, to drive cellular apoptosis owing to broken dsDNA. Furthermore, translated viral proteins, such Vank86, 92, and 101, known to complex with transcription factor Dip3, positively regulated DHYS and DOHH transcription maintaining the activation of the eIF5A-hypusination. Mechanistically, MbBV-mediated extracellular vesicles contained inserted viral fragments that re-integrated into recipients, potentially via the homologous recombinant repair system. Meanwhile, this stimulation regulated activated caspase-3 levels via PI3K/AKT 308 and 473 dephosphorylation to promote apoptosis of granulocyte-like recipients Sf9 cell; maintaining PI3K/AKT 473 phosphorylation and 308 dephosphorylation inhibited caspase-3 activation leading to dismigration of plasmatocyte-like recipient High Five cells. Together, our results suggest that integration-mediated eIF5A hypusination drives extracellular vesicles for continuous immunosuppression.

Lymphatic and Blood Endothelial Extracellular Vesicles: A Story Yet to Be Written.

Extracellular vesicles (EVs), such as exosomes, microvesicles, and apoptotic bodies, are cell-derived, lipid bilayer-enclosed particles mediating intercellular communication and are therefore vital for transmitting a plethora of biological signals. The vascular endothelium substantially contributes to the circulating particulate secretome, targeting important signaling pathways that affect blood cells and regulate adaptation and plasticity of endothelial cells in a paracrine manner. Different molecular signatures and functional properties of endothelial cells reflect their heterogeneity among different vascular beds and drive current research to understand varying physiological and pathological effects of blood and lymphatic endothelial EVs. Endothelial EVs have been linked to the development and progression of various vascular diseases, thus having the potential to serve as biomarkers and clinical treatment targets. This review aims to provide a brief overview of the human vasculature, the biology of extracellular vesicles, and the current knowledge of endothelium-derived EVs, including their potential role as biomarkers in disease development.

Extracellular Vesicles and Their Emerging Roles as Cellular Messengers in Endocrinology: An Endocrine Society Scientific Statement.

During the last decade, there has been great interest in elucidating the biological role of extracellular vesicles (EVs), particularly, their hormone-like role in cell-to-cell communication. The field of endocrinology is uniquely placed to provide insight into the functions of EVs, which are secreted from all cells into biological fluids and carry endocrine signals to engage in paracellular and distal interactions. EVs are a heterogeneous population of membrane-bound vesicles of varying size, content, and bioactivity. EVs are specifically packaged with signaling molecules, including lipids, proteins, and nucleic acids, and are released via exocytosis into biofluid compartments. EVs regulate the activity of both proximal and distal target cells, including translational activity, metabolism, growth, and development. As such, EVs signaling represents an integral pathway mediating intercellular communication. Moreover, as the content of EVs is cell-type specific, it is a "fingerprint" of the releasing cell and its metabolic status. Recently, changes in the profile of EV and bioactivity have been described in several endocrine-related conditions including diabetes, obesity, cardiovascular diseases, and cancer. The goal of this statement is to highlight relevant aspects of EV research and their potential role in the field of endocrinology.

Isolation and Fluorescent Labeling of Extracellular Vesicles from Cultured Tumor Cells.

Extracellular vesicles (EVs), comprising exosomes, ectosomes, and apoptotic bodies, are an important component of molecular cell-to-cell communication, and are critically involved in the pathophysiology of various diseases, including tumors. In order to study the interaction of tumor cell-derived EVs with their target cells and to investigate their biological functions in comparison to other tumor cell-released factors, efficient isolation of EVs from cultured tumor cells, as well as fluorescent labeling of these EVs, is often necessary. In addition, EVs and EV-like particles are emerging as versatile vehicles for the delivery of therapeutic substances. Here, we describe an easy size exclusion chromatography-based method to isolate EVs from the mouse melanoma cell line B16F10 that yields highly enriched EV samples for subsequent applications such as molecular and functional studies. Our protocol also includes an optional labeling step with the lipophilic dye DiD, which allows tracking of EV uptake by recipient cells in vitro and in vivo.

Apoptotic Body-Mediated Intracellular Delivery Strategy for Enhanced STING Activation and Improved Tumor Immunogenicity.

Agonists of stimulators of interferon genes (STING) are a promising class of immunotherapeutics that trigger potent innate immunity. However, the therapeutic efficacy of conventional STING agonists, such as 2',3'-cyclic guanosine monophosphate-adenosine monophosphate (cGAMP), is severely restricted to poor cytosolic delivery and lacks the capacity to promote the recognition of tumor-specific antigens. Here, we tackle these challenges through a nanovaccine platform based on Fenton-reactive and STING-activating nanoparticles, synergistically contributing to the generation of tumor-cell-derived apoptotic bodies (ABs). ABs loaded with exogenous cGAMP are readily phagocytosed by antigen-presenting cells (APCs), as a Trojan horse for rendering tumor cells with high immunogenicity instead of a noninflammatory response. This leads to enhanced STING activation and an improved tumor-specific antigen presentation ability, boosting the adaptive immunity in collaboration with innate immune. The strategy of exploiting a metal-based nanovaccine platform possesses great potential to be clinically translated into a trinitarian system of diagnosis, treatment, and prognosis.

Accelerated co-cultured dendritic cell (acDC) loaded with autologous apoptotic bodies might be a promising approach for antigen delivery.

Antigens derived from engulfed apoptotic bodies that are presented by dendritic cells can amplify Ag-specific T-cells. Accelerated co-cultured DC (acDC) strategy keeps lymphocytes in contact with differentiating DCs. Therefore, Ag-specific T-cell activation can occur during DC maturation. Our aim was to prepare DCs by acDC method and check the subsequent engulfment of the apoptotic body by acDC. We have proposed that this method could be feasible if we transfect the apoptotic bodies with the antigen. DCs were prepared using acDC method and their maturation markers were confirmed by flow cytometry. Ultraviolet was used for inducing apoptosis in the PBMCs and induction of apoptosis checked by propidium iodide and 7-aminoactinomycin D staining. Flow cytometry and immunohistochemistry were used for checking the uptake of apoptotic bodies by the DCs. The alloreactivity against apoptotic bodies was examined by enzyme-linked immunospot (ELISPOT) assay. Results showed that 40.4% of DCs could efficiently engulf the apoptotic bodies. The results indicated that acDC method is capable to isolate a high yield of DCs, and these cells could properly engulf the apoptotic bodies, more works should be performed to use this method for Ag discovery through delivering the Ag by apoptotic bodies into the DCs.

Biodistribution of extracellular vesicles following administration into animals: A systematic review.

In recent years, attention has turned to examining the biodistribution of EVs in recipient animals to bridge between knowledge of EV function in vitro and in vivo. We undertook a systematic review of the literature to summarize the biodistribution of EVs following administration into animals. There were time-dependent changes in the biodistribution of small-EVs which were most abundant in the liver. Detection peaked in the liver and kidney in the first hour after administration, while distribution to the lungs and spleen peaked between 2-12 h. Large-EVs were most abundant in the lungs with localization peaking in the first hour following administration and decreased between 2-12 h. In contrast, large-EV localization to the liver increased as the levels in the lungs decreased. There was moderate to low localization of large-EVs to the kidneys while localization to the spleen was typically low. Regardless of the origin or size of the EVs or the recipient species into which the EVs were administered, the biodistribution of the EVs was largely to the liver, lungs, kidneys, and spleen. There was extreme variability in the methodology between studies and we recommend that guidelines should be developed to promote standardization where possible of future EV biodistribution studies.

Overview and Update on Methods for Cargo Loading into Extracellular Vesicles.

The enormous library of pharmaceutical compounds presents endless research avenues. However, several factors limit the therapeutic potential of these drugs, such as drug resistance, stability, off-target toxicity, and inadequate delivery to the site of action. Extracellular vesicles (EVs) are lipid bilayer-delimited particles and are naturally released from cells. Growing evidence shows that EVs have great potential to serve as effective drug carriers. Since EVs can not only transfer biological information, but also effectively deliver hydrophobic drugs into cells, the application of EVs as a novel drug delivery system has attracted considerable scientific interest. Recently, EVs loaded with siRNA, miRNA, mRNA, CRISPR/Cas9, proteins, or therapeutic drugs show improved delivery efficiency and drug effect. In this review, we summarize the methods used for the cargo loading into EVs, including siRNA, miRNA, mRNA, CRISPR/Cas9, proteins, and therapeutic drugs. Furthermore, we also include the recent advance in engineered EVs for drug delivery. Finally, both advantages and challenges of EVs as a new drug delivery system are discussed. Here, we encourage researchers to further develop convenient and reliable loading methods for the potential clinical applications of EVs as drug carriers in the future.

Apoptotic Bodies: Mechanism of Formation, Isolation and Functional Relevance.

In the final stages of apoptosis, apoptotic cells can generate a variety of membrane-bound vesicles known as apoptotic extracellular vesicles (ApoEVs). Apoptotic bodies (ApoBDs), a major subset of ApoEVs, are formed through a process termed apoptotic cell disassembly characterised by a series of tightly regulated morphological steps including plasma membrane blebbing, apoptotic membrane protrusion formation and fragmentation into ApoBDs. To better characterise the properties of ApoBDs and elucidate their function, a number of methods including differential centrifugation, filtration and fluorescence-activated cell sorting were developed to isolate ApoBDs. Furthermore, it has become increasingly clear that ApoBD formation can contribute to various biological processes such as apoptotic cell clearance and intercellular communication. Together, recent literature demonstrates that apoptotic cell disassembly and thus, ApoBD formation, is an important process downstream of apoptotic cell death. In this chapter, we discuss the current understandings of the molecular mechanisms involved in regulating apoptotic cell disassembly, techniques for ApoBD isolation, and the functional roles of ApoBDs in physiological and pathological settings.