Publications

Background: Tyrosine kinase inhibitors (TKI) were initially demonstrated as an efficacious treatment for renal cell carcinoma (RCC). However, after a median treatment length of 14 months, a vast majority of patients develop resistance. This study analyzed a combination therapy of tipifarnib (Tipi) + sunitinib that targeted exosome-conferred drug resistance. Methods: 786-O, 786-O-SR (sunitinib resistant), A498, A498-SR, Caki-2, Caki-2-SR, and 293T cells were cultured. Exosomes were collected using differential ultracentrifugation. Cell proliferation, Jurkat T cell immune assay, and immunoblot analysis were used for downstream analysis. Results: SR exosomes treatment displayed a cytotoxic effect on immune cells. This cytotoxic effect was associated with increased expression of PD-L1 on SR exosomes when compared to sunitinib-sensitive (SS) exosomes. Additionally, Tipi treatment downregulated PD-L1 expression on exosomes derived from SR cell lines. Tipi’s ability to downregulate PD-L1 in exosomes has a significant application within patients. Exosomes collected from patients with RCC showed increased PD-L1 expression over subjects without RCC. Next, exosome concentrations were then compared after Tipi treatment, with all SS cell lines displaying an even greater reduction. On immunoblot assay, 293T cells showed a dose-dependent increase in Alix with no change in either nSMase or Rab27a. Conversely, all the SS and SR cell lines displayed a decrease in all three markers. After a cell proliferation employed a 48-h treatment on all SS and SR cell lines, the drug combination displayed synergistic ability to decrease tumor growth. Conclusions: Tipifarnib attenuates both the exosome endosomal sorting complex required for endosomal sorting complex required for transport (ESCRT)-dependent and ESCRT-independent pathways, thereby blocking exosome biogenesis and secretion as well as downregulating PD-L1 on SS and SR cells.

One of the largest surfaces of the human body in contact with the environment is the respiratory epithelium, constituted by different specialized cells. Alveolar type I cells are involved in gas exchange whereas alveolar type II cells prevent the alveoli from collapsing due to the synthesis and secretion of lung surfactant (LS). This lipid-protein complex covers the alveolar surface and reduces surface tension at the air-liquid interface. LS, the first element in contact with inhaled air, is also involved in innate defense mechanisms. Surfactant protein C (SP-C) is a small hydrophobic transmembrane protein crucial for the biophysical function of LS. Different studies have revealed that the palmitoylation state of SP-C modulates important protein-lipid interactions within surfactant layers. Moreover, recent research has revealed that SP-C oligomerization, presumably through two structural motifs in SP-C sequence, could promote membrane fragmentation and enhance membrane vesicle alveolar uptake highlighting a key potential role of SP-C in LS homeostasis. In this work, we have analyzed the effect of palmitoylation on SP-C-promoted membrane fragmentation and vesicle uptake in the LS context. To do so, we have compared the behavior in different assays of the native palmitoylated protein and a recombinant SP-C version lacking palmitoyl chains, once reconstituted in two different lipid models mimicking LS membranes. Likewise, we have studied the implication of the proposed dimerization motifs in the SP-C sequence by testing synthetic peptides with selected sequence variations. Results from tunable resistive pulse sensing experiments suggest that both palmitoylation and the oligomerization state of SP-C are important to promote fission of membranes. Protein oligomerization and membrane fragmentation have been also analyzed with respect to membrane vesicle internalization by alveolar-derived cell lines, as evaluated by flow cytometry of cell cultures exposed to fluorescent lipid/protein complexes.

The constant dialogue between the plant world and the animal world (including man among them) has been known since the time of Adam and Eve, where an apple was the origin of the evils of the world. Apart from Snow White-who might have something to object to when it comes to the use of apples-fruits, plants, and natural extracts have been known for millennia as remedies for human health-related ailments. In the light of such evidence, the aim of the present work was to investigate from a biological point of view the potential role of apple exosomes in inflammatory processes on human cells. To this end we isolated and characterized apple exosomes and treated human cells such as macrophages and NCTC L929 as cancer cells in order to evaluate the tumorigenic and anti-inflammatory effect of apple exomes. Microscopic and molecular biology analyses were conducted to characterize exosomes and to assess cell proliferation, death, and miRNA line, as well as gene expression and the uptake of exosomes by cells. The results confirm the absolute biological safety of exosomes and their anti-inflammatory effect, mediated mainly by miRNA146 production by M2 macrophages.

The constant dialogue between the plant world and the animal world (including man among them) has been known since the time of Adam and Eve, where an apple was the origin of the evils of the world. Apart from Snow White—who might have something to object to when it comes to the use of apples—fruits, plants, and natural extracts have been known for millennia as remedies for human health-related ailments. In the light of such evidence, the aim of the present work was to investigate from a biological point of view the potential role of apple exosomes in inflammatory processes on human cells. To this end we isolated and characterized apple exosomes and treated human cells such as macrophages and NCTC L929 as cancer cells in order to evaluate the tumorigenic and anti-inflammatory effect of apple exomes. Microscopic and molecular biology analyses were conducted to characterize exosomes and to assess cell proliferation, death, and miRNA line, as well as gene expression and the uptake of exosomes by cells. The results confirm the absolute biological safety of exosomes and their anti-inflammatory effect, mediated mainly by miRNA146 production by M2 macrophages.

Nanoparticles can acquire a plasma protein corona defining their biological identity. Corona functions were previously considered for cell-derived extracellular vesicles (EVs). Here we demonstrate that nano-sized EVs from therapy-grade human placental-expanded (PLX) stromal cells are surrounded by an imageable and functional protein corona when enriched with permissive technology. Scalable EV separation from cell-secreted soluble factors via tangential flow-filtration (TFF) and subtractive tandem mass-tag (TMT) proteomics revealed significant enrichment of predominantly immunomodulatory and proangiogenic proteins. Western blot, calceinbased flow cytometry, super-resolution and electron microscopy verified EV identity. PLX-EVs partly protected corona proteins from protease digestion. EVs significantly ameliorated human skin regeneration and angiogenesis in vivo, induced differential signalling in immune cells, and dose-dependently inhibited T cell proliferation in vitro. Corona removal by size-exclusion or ultracentrifugation abrogated angiogenesis. Re-establishing an artificial corona by cloaking EVs with fluorescent albumin as a model protein or defined proangiogenic factors was depicted by superresolution microscopy, electron microscopy and zeta-potential shift, and served as a proof-of-concept. Understanding EV corona formation will improve rational EVinspired nano-therapy design.

Background Extracellular vesicles (EVs) are produced by all cell types and serve as biological packets delivering a wide variety of molecules for cell-to-cell communication. However, the biology of the EV extravesicular surface domain that we have termed EV ‘biocorona’ remains underexplored. Upon cell secretion, EVs possess an innate biocorona containing membrane integral and peripheral constituents that is modified by acquired constituents post secretion. This distinguishes EVs from synthetic nanoparticulate biomaterials that are limited to an adsorption-based, acquired biocorona. Methods The EV biocorona molecular constituents were radiolabeled with 125I to study biocorona constituents and its surface dynamics. As example toolset applications, 125I-EVs were utilized to study EV cell trafficking and the stability of the EV biocorona during storage. Results The biocorona of EVs consisted of proteins, lipids, DNA and RNA. The cellular uptake of 125I-EVs was temperature dependent and internalized 125I-EVs were rapidly recycled by cells. When 125I-EVs were stored in a purified state, they exhibited time and temperature dependent biocorona shedding and proteolytic degradation that was partially inhibited in the presence of serum. Conclusion The EV biocorona is complex and dynamic. Radiolabeling of the EV biocorona enables a unique platform methodology to study the biocorona and will facilitate unlocking EV's full clinical translation potential. General significance The EV biocorona affects EV mediated biological processes in health and disease. Acquiring knowledge of the EV biocorona composition, dynamics, stability and structure not only informs the diagnostic and therapeutic translation of EVs but also aids in designing biomimetic nanomaterials for drug delivery.

Nanoscale extracellular vesicles (EVs) represent a unique cellular derivative that reflect the therapeutic potential of mesenchymal stem cells (MSCs) toward tissue engineering and injury repair without the logistical and safety concerns of utilizing living cells. However, upon systemic administration in vivo,EVs undergo rapid clearance and typically lack controlled targeted delivery, thus reducing their effectiveness in therapeutic regenerative therapies. Here, we describe a strategy that enables long-term in vivo spatial EV retention by chemoselective immobilization of metabolically incoporated azido ligand-bearing EVs (azido-EVs) within a dibenzocyclooctyne-modified collagen hydrogel. MSC-derived azido-EVs exhibit comparable morphological and functional properties as their non-labeled EV counterparts and, when immobilized within collagen hydrogel implants via click chemistry, they elicited more robust host cell infiltration, angiogenic and immunoregulatory responses including vascular ingrowth and macrophage recruitment compared to ten times the higher dose required by non-immobilized EVs. We envision this technology will enable a wide range of applications to spatially promote vascularization and host integration relevant to tissue engineering and regenerative medicine applications.

Androgenetic alopecia (AGA) is the most common type of hair loss in men and women. Dihydrotestosterone (DHT) and androgen receptor (AR) levels are increased in patients with AGA, and DHT-AR signaling correlates strongly with AGA pathogenesis. In this study, treatment with self-assembled micelle inhibitory RNA (SAMiRNA) nanoparticle-type siRNA selectively suppressed AR expression in vitro. Clinical studies with application of SAMiRNA to the scalp and massaging to deliver it to the hair follicle confirmed its efficacy in AGA. For identification of a potent SAMiRNA for AR silencing, 547 SAMiRNA candidates were synthesized and screened. SAMiRNA-AR68 (AR68) was the most potent and could be efficiently delivered to human follicle dermal papilla cells (HFDPCs) and hair follicles, and this treatment decreased the AR mRNA and protein levels. We confirmed that 10 µM AR68 elicits no innate immune response in human PBMCs and no cytotoxicity up to 20 µM with HFDP and HaCaT cells. Clinical studies were performed in a randomized and double-blind manner with two different doses and frequencies. In the low-dose (0.5 mg/ml) clinical study, AR68 was applied three times per week for 24 weeks, and through quantitative analysis using a phototrichogram, we confirmed increases in total hair counts. In the high-dose (5 mg/ml) clinical study, AR68 was given once per week for 24 weeks and showed 83% efficacy in increasing hair counts compared with finasteride. No side effects were observed. Therefore, SAMiRNA targeting AR mRNA is a potential novel topical treatment for AGA.

Abnormal cellular lipid metabolism has a very important role in the occurrence and progression of diabetic kidney disease (DKD). However, the lipid composition and differential expression by high glucose stimulation of renal tubular cells and their exosomes, which is a vital part of the development of DKD, are largely unknown. In this study, based on targeted lipid analysis by isotope labeling and tandem mass spectrometry, a total of 421 and 218 lipid species were quantified in HK-2 cells and exosomes, respectively. More importantly, results showed that GM3 d18:1/22:0, GM3 d18:1/16:0, GM3 d18:0/16:0, GM3 d18:1/22:1 were significantly increased, while LPE18:1, LPE, CL66:4 (16:1), BMP36:3, CL70:7 (16:1), CL74:8 (16:1) were significantly decreased in high glucose-stimulated HK-2 cells. Also, PG36:1, FFA22:5, PC38:3, SM d18:1/16:1, CE-16:1, CE-18:3, CE-20:5, and CE-22:6 were significantly increased, while GM3 d18:1/24:1, GM3 were significantly decreased in exosomes secreted by high glucose-stimulated HK-2 cells. Furthermore, TAG, PC, CL were decreased significantly in the exosomes comparing with the HK-2 cells, and LPA18:2, LPI22:5, PG32:2, FFA16:1, GM3 d18:1/18:1, GM3 d18:1/20:1, GM3 d18:0/20:0, PC40:6p, TAG52:1(18:1), TAG52:0(18:0), CE-20:5, CE-20:4, CE-22:6 were only found in exosomes. In addition, the expression of PI4P in HK-2 cells decreased under a high glucose state. These data may be useful to provide new targets for exploring the mechanisms of DKD.

Within this study, the performance and limitations of tunable resistive pulse sensing (TRPS) was evaluated to characterize submicron particles in unstressed and heat stressed monoclonal antibody (mAb) solutions. These were compared with microfluidic resistive pulse sensing (MRPS), resonant mass measurement (RMM), and nanoparticle tracking analysis (NTA). For TRPS and MRPS measurements, an adjustment of ionic strength was required to achieve suitable measurement conditions. The addition of electrolytes is potentially critical for protein formulations and therefore the effect of salt concentration and pH on submicron particle levels was further investigated. Heat stress caused a sharp increase in particle levels between 250-900 nm, observable by all four techniques. Due to reduced colloidal stability, indicated by increased attractive forces and reduced aggregation onset temperatures in the presence of sodium chloride, protein aggregation was observed in heat stressed mAb only after the addition of sodium chloride. Achieving adequate ionic strength by replacing sodium chloride with other electrolytes similarly resulted in reduced colloidal stability and protein aggregation. It is recommended that protein samples prone for aggregation in the presence of high ionic strength should not be analyzed by RPS measurements after the addition of electrolytes. However, protein samples containing already required ionic strength can be analyzed by any of the four techniques.

Small extracellular vesicles (sEVs) secreted from cancer cells play pivotal roles in cancer metastasis and malignancy by transferring biomolecules and conditioning future metastatic sites. Studies have elucidated structures and functions of glycans on sEVs; however, whether sEVs remodel glycans in recipient cells remains poorly understood. Here, we examined the enzyme activity of glycosyltransferases for complex N-glycan biosynthesis in cancer-derived sEVs and discovered that cancer-related glycosyltransferase, N-acetylglucosaminyltransferase-V (GnT-V, a.k.a. MGAT5), is selectively enriched in sEVs among various glycosyltransferases. GnT-V in sEVs is a cleaved form, and cleavage by SPPL3 protease is necessary for loading GnT-V in sEVs. Fractionation experiments and single-particle imaging further revealed that GnT-V was enriched in non-exosomal sEVs. Strikingly, we found that enzymatically active GnT-V in sEVs was transferred to recipient cells and the N-glycan structures of recipient cells were remodeled to express GnT-V-produced glycans. Our results suggest GnT-V-enriched sEVs’ role in glycan remodeling in cancer metastasis.

We present an innovative approach allowing the identification, isolation, and molecular characterization of disease-related exosomes based on their different antigenic reactivities. The designed strategy could be immediately translated into any disease in which exosomes are involved. The identification of specific markers and their subsequent association with exosome subtypes, together with the possibility to engineer target-guided exosome-like particles, could represent the key for the effective adoption of exosomes in clinical practice.

Immune-inflammatory activation impacts extracellular vesicles (EVs), including their miRNA cargo. There is evidence for changes in the EV miRNome in inflammation-associated neuropsychiatric disorders. This mouse study investigated: (1) effects of systemic lipopolysaccharide (LPS) and chronic social stress (CSS) on plasma EV miRNome; and (2) physiological, transcriptional, and behavioural effects of peripheral or central delivered LPS-activated EVs in recipient mice. LPS or CSS effects on the plasma EV miRNome were assessed by using microRNA sequencing. Recipient mice received plasma EVs isolated from LPS-treated or SAL-treated donor mice or vehicle only, either intravenously or into the nucleus accumbens (NAc), on three consecutive days. Bodyweight, spleen or NAc transcriptome and reward (sucrose) motivation were assessed. LPS and CSS increased the expression of 122 and decreased expression of 20 plasma EV miRNAs, respectively. Peripheral LPS-EVs reduced bodyweight, and both LPS-EVs and SAL-EVs increased spleen expression of immune-relevant genes. NAc-infused LPS-EVs increased the expression of 10 immune-inflammatory genes. Whereas motivation increased similarly across test days in all groups, the effect of test days was more pronounced in mice that received peripheral or central LPS-EVs compared with other groups. This study provides causal evidence that increased EV levels impact physiological and behavioural processes and are of potential relevance to neuropsychiatric disorders.

Acoustofluidicly manipulated microbubbles (MBs) and echogenic liposomes (ELIPs) have been suggested as drug delivery systems for the 'on demand' release of drug in target tissue. This requires a clear understanding of their behaviour during ultrasonication and after ultrasonication stops. The main focus of this study is to investigate the behaviour of MBs and ELIPs clusters after ultrasonication stops and the underlaying cause of cluster diffusion considering electrostatic repulsion, steric repulsion and Brownian motion. It also examines the capability of existing models used to predict MBs' attraction velocity due to secondary radiation force, on predicting ELIPs' attraction velocity. Tunable resistive pulse sensing (TRPS) and phase analysis light scattering (PALS) techniques were used to measure zeta potentials of the agents and the size distributions were measured using TRPS. The zeta potentials were found to be -2.43 mV and -0.62 mV for Definity™ MBs, and -3.62 mV and -2.35 mV for ELIPs using TRPS and PALS, respectively. Both agents were shown to have significant cluster formation at pressures as low as 6 kPa. Clusters of both agents were shown to diffuse as sonication stops at a rate that approximately equals the sum of the diffusion coefficients of the agents forming them. The de-clustering behaviours are due to Brownian motion as no sign of electrostatic repulsion was observed and particles movements were observed to be faster for smaller diameters. These findings are important to design and optimise effective drug delivery systems using acoustofluidically manipulated MBs and ELIPs.

Non-coding RNA (ncRNA), released into circulation or packaged into exosomes, plays important roles in many biological processes in the kidney. The purpose of the present study is to identify a common ncRNA signature associated with early renal damage and its related molecular pathways. Three individual libraries (plasma and urinary exosomes, and total plasma) were prepared from each hypertensive patient (with or without albuminuria) for ncRNA sequencing analysis. Next, an RNA-based transcriptional regulatory network was constructed. The three RNA biotypes with the greatest number of differentially expressed transcripts were long-ncRNA (lncRNA), microRNA (miRNA) and piwi-interacting RNA (piRNAs). We identified a common 24 ncRNA molecular signature related to hypertension-associated urinary albumin excretion, of which lncRNAs were the most representative. In addition, the transcriptional regulatory network showed five lncRNAs (LINC02614, BAALC-AS1, FAM230B, LOC100505824 and LINC01484) and the miR-301a-3p to play a significant role in network organization and targeting critical pathways regulating filtration barrier integrity and tubule reabsorption. Our study found an ncRNA profile associated with albuminuria, independent of biofluid origin (urine or plasma, circulating or in exosomes) that identifies a handful of potential targets, which may be utilized to study mechanisms of albuminuria and cardiovascular damage.

Screening, monitoring, and diagnosis are critical in oncology treatment. However, there are limitations with the current clinical methods, notably the time, cost, and special facilities required for radioisotope-based methods. An alternative approach, which uses magnetic beads, offers faster analyses with safer materials over a wide range of oncological applications. Magnetic beads have been used to detect extracellular vesicles (EVs) in the serum of pancreatic cancer patients with statistically different EV levels in preoperative, postoperative, and negative control samples. By incorporating fluorescence, magnetic beads have been used to quantitatively measure prostate-specific antigen (PSA), a prostate cancer biomarker, which is sensitive enough even at levels found in healthy patients. Immunostaining has also been incorporated with magnetic beads and compared with conventional immunohistochemical methods to detect lesions; the results suggest that immunostained magnetic beads could be used for pathological diagnosis during surgery. Furthermore, magnetic nanoparticles, such as superparamagnetic iron oxide nanoparticles (SPIONs), can detect sentinel lymph nodes in breast cancer in a clinical setting, as well as those in gallbladder cancer in animal models, in a surgery-applicable timeframe. Ultimately, recent research into the applications of magnetic beads in oncology suggests that the screening, monitoring, and diagnosis of cancers could be improved and made more accessible through the adoption of this technology.

The in vivo function of cell-derived extracellular vesicles (EVs) is challenging to establish since cell-specific EVs are difficult to isolate and differentiate. We, therefore, created an EV reporter using truncated CD9 to display enhanced green fluorescent protein (EGFP) on the EV surface. CD9truc-EGFP expression in cells did not affect EV size and concentration but enabled co-precipitation of EV markers TSG101 and ALIX from the cell-conditioned medium by anti-GFP immunoprecipitation. We then created a transgenic mouse where CD9truc-EGFP was inserted in the inverse orientation and double-floxed, ensuring irreversible Cre recombinase-dependent EV reporter expression. We crossed the EV reporter mice with mice expressing Cre ubiquitously (CMV-Cre), in cardiomyocytes (αMHC-MerCreMer) and renal tubular epithelial cells (Pax8-Cre), respectively. The CD9truc-EGFP positive mice showed Cre-dependent EGFP expression, and plasma CD9truc-EGFP EVs were immunoprecipitated only from CD9truc-EGFP positive CD9truc-EGFPxCMV-Cre and CD9truc-EGFPxαMHC-Cre mice, but not in CD9truc-EGFPxPax8-Cre and CD9truc-EGFP negative mice. In urine samples, CD9truc-EGFP EVs were detected by immunoprecipitation only in CD9truc-EGFP positive CD9truc-EGFPxCMV-Cre and CD9truc-EGFPxPax8-Cre mice, but not CD9truc-EGFPxαMHC-Cre and CD9truc-EGFP negative mice. In conclusion, our EV reporter mouse model enables Cre-dependent EV labeling, providing a new approach to studying cell-specific EVs in vivo and gaining a unique insight into their physiological and pathophysiological function.

Non-small-cell lung cancer (NSCLC) is defined as the most universally diagnosed class of lung cancer. Cisplatin (DDP) is an effective drug for NSCLC, but tumors are prone to drug resistance. The current study set out to evaluate the regulatory effect of long non-coding RNA (lncRNA) small nucleolar RNA host gene 12 (SNHG12) in extracellular vesicles (EVs) derived from carcinoma-associated fibroblasts (CAFs) on DDP resistance in NSCLC cells. Firstly, NSCLC cells were treated with EVs, followed by detection of cell activity, IC50 values, cell proliferation and apoptosis, and Cy3-SNHG12. We observed that CAFs-EVs promoted IC50 values and cell proliferation and inhibited apoptosis. In addition, we learned that lncRNA SNHG12 carried by CAFs-EVs into NSCLC facilitated DDP resistance of NSCLC cells. Furthermore, ELAV like RNA binding protein 1 (HuR/ELAVL1) binding to lncRNA SNHG12 and X-linked inhibitor of apoptosis (XIAP) was verified and RNA stability of XIAP was also verified CAFs-EVs promoted RNA stability and transcription of XIAP, while silencing HuR could partially-reverse this promoting effect. Further joint experimentation showed that silencing XIAP partially inhibited DDP resistance in NSCLC cells. Additionally, the tumor growth and the positive rate of Ki67 and HuR were detected, which showed that CAFs-oe-EVs promoted the tumor and the positive rate of Ki67, as well as the levels of lncRNA SNHG12, HuR, and XIAP in vivo. Collectively, our findings indicated that lncRNA SNHG12 carried by CAFs-EVs into NSCLC cells promoted RNA stability and XIAP transcription by binding to HuR, thus augmenting DDP resistance in NSCLC cells.

Background Breast cancer is the most common cancer, and the leading cause of cancer-related deaths, among females world-wide. Recent research suggests that extracellular vesicles (EVs) play a major role in the development of breast cancer metastasis. Axillary lymph node dissection (ALND) is a procedure in patients with known lymph node metastases, and after surgery large amounts of serous fluid are produced from the axilla. The overall aim was to isolate and characterize EVs from axillary serous fluid, and more specifically to determine if potential breast cancer biomarkers could be identified. Methods Lymphatic drain fluid was collected from 7 patients with breast cancer the day after ALND. EVs were isolated using size exclusion chromatography, quantified and detected by nanoparticle tracking analysis, electron microscopy, nano flow cytometry and western blot. The expression of 37 EV surface proteins was evaluated by flow cytometry using the MACSPlex Exosome kit. Results Lymphatic drainage exudate retrieved after surgery from all 7 patients contained EVs. The isolated EVs were positive for the typical EV markers CD9, CD63, CD81 and Flotillin-1 while albumin was absent, indicating low contamination from blood proteins. In total, 24 different EV surface proteins were detected. Eleven of those proteins were detected in all patients, including the common EV markers CD9, CD63 and CD81, cancer-related markers CD24, CD29, CD44 and CD146, platelet markers CD41b, CD42a and CD62p as well as HLA-DR/DP/DQ. Furthermore, CD29 and CD146 were enriched in Her2+ patients compared to patients with Her2- tumors. Conclusions Lymphatic drainage exudate retrieved from breast cancer patients after surgery contains EVs that can be isolated using SEC isolation. The EVs have several cancer-related markers including CD24, CD29, CD44 and CD146, proteins of potential interest as biomarkers as well as to increase the understanding of the mechanisms of cancer biology.

Ionizing radiation (IR)-induced bystander effects contribute to biological responses to radiation, and extracellular vesicles (EVs) play important roles in mediating these effects. In this study we investigated the role of bone marrow (BM)-derived EVs in the bystander transfer of radiation damage. Mice were irradiated with 0.1Gy, 0.25Gy and 2Gy, EVs were extracted from the BM supernatant 24 h or 3 months after irradiation and injected into bystander mice. Acute effects on directly irradiated or EV-treated mice were investigated after 4 and 24 h, while late effects were investigated 3 months after treatment. The acute effects of EVs on the hematopoietic stem and progenitor cell pools were similar to direct irradiation effects and persisted for up to 3 months, with the hematopoietic stem cells showing the strongest bystander responses. EVs isolated 3 months after irradiation elicited no bystander responses. The level of seven microRNAs (miR-33a-3p, miR-140-3p, miR-152-3p, miR-199a-5p, miR-200c-5p, miR-375-3p and miR-669o-5p) was altered in the EVs isolated 24 hour but not 3 months after irradiation. They regulated pathways highly relevant for the cellular response to IR, indicating their role in EV-mediated bystander responses. In conclusion, we showed that only EVs from an early stage of radiation damage could transmit IR-induced bystander effects.

Extracellular Vesicles and Circulating Nucleic Acids is an open access journal, focusing on extracellular vesicles and circulating nucleic acids including DNA, RNA, and miRNA and their therapeutic use.

Tumor peptides associated with MHC class I molecules or their synthetic variants have attracted great attention for their potential use as vaccines to induce tumor-specific CTLs. However, the outcome of clinical trials of peptide-based tumor vaccines has been disappointing. There are various reasons for this lack of success, such as difficulties in delivering the peptides specifically to professional Ag-presenting cells, short peptide half-life in vivo, and limited peptide immunogenicity. We report here a novel peptide vaccination strategy that efficiently induces peptide-specific CTLs. Nanoparticles (NPs) were fabricated from a biodegradable polymer, poly(D,L-lactic-co-glycolic acid), attached to H-2Kb molecules, and then the natural peptide epitopes associated with the H-2Kb molecules were exchanged with a model tumor peptide, SIINFEKL (OVA257-268). These NPs were efficiently phagocytosed by immature dendritic cells (DCs), inducing DC maturation and activation. In addition, the DCs that phagocytosed SIINFEKL-pulsed NPs potently activated SIINFEKL-H-2Kb complex-specific CD8+ T cells via cross-presentation of SIINFEKL. In vivo studies showed that intravenous administration of SIINFEKL-pulsed NPs effectively generated SIINFEKL-specific CD8+ T cells in both normal and tumor-bearing mice. Furthermore, intravenous administration of SIINFEKL-pulsed NPs into EG7.OVA tumor-bearing mice almost completely inhibited the tumor growth. These results demonstrate that vaccination with polymeric NPs coated with tumor peptide-MHC-I complexes is a novel strategy for efficient induction of tumor-specific CTLs.

Testicular endothelial cells have been found to play an important role in spermatogenesis and fertility, but their mechanism is obscure. Exosomes released by various cells are recognized as cell-cell communication mediators during the initiation and progression of many diseases. Therefore, the current study aimed to investigate the protein and miRNA components of human testicular endothelial cell-derived exosomes (HTEC-Exos) and to explore their potential effects on spermatogenesis. In this study, HTEC-Exos were first isolated by the ultracentrifugation method, and then identified by nanoparticle tracking analysis, transmission electron microscopy (TEM), and western blotting. The characteristics of HTEC-Exos were examined by liquid chromatography-mass spectrometry and microRNA (miRNA) chip analysis. Bioinformatics analysis was performed to explore the potential role of the exosomal content on spermatogenesis. A total of 945 proteins were identified, 11 of which were closely related to spermatogenesis. A total of 2578 miRNAs were identified. Among them, 30 miRNAs demonstrated potential associations with male reproductive disorders, such as azoospermia, and spermatogenesis disorders. In particular, 11 out of these 30 miRNAs have been proven to be involved in spermatogenesis based on available evidence. This study provides a global view of the proteins and miRNAs from HTEC-Exos, suggesting that HTEC-Exos may function as potential effectors during the process of spermatogenesis.

Myotonic dystrophy type 1 (DM1) is the most common adult-onset muscular dystrophy, primarily characterized by muscle wasting and weakness. Many biomarkers already exist in the rapidly developing biomarker research field that aim to improve patients’ care. Limited work, however, has been performed on rare diseases, including DM1. We have previously shown that specific microRNAs (miRNAs) can be used as potential biomarkers for DM1 progression. In this report, we aimed to identify novel serum-based biomarkers for DM1 through high-throughput next-generation sequencing. A number of miRNAs were identified that are able to distinguish DM1 patients from healthy individuals. Two miRNAs were selected, and their association with the disease was validated in a larger panel of patients. Further investigation of miR-223-3p, miR-24-3p, and the four previously identified miRNAs, miR-1-3p, miR-133a-3p, miR-133b-3p, and miR-206-3p, showed elevated levels in a DM1 mouse model for all six miRNAs circulating in the serum compared to healthy controls. Importantly, the levels of miR-223-3p, but not the other five miRNAs, were found to be significantly downregulated in five skeletal muscles and heart tissues of DM1 mice compared to controls. This result provides significant evidence for its involvement in disease manifestation.

Hydroxyapatite (HA), as the main mineral component in hard tissues, has good biocompatibility. In particular, HA films are widely used as bioactive coatings for artificial bones and dental implants in biomedical fields. However, it is currently difficult to prepare a nanostructure-controlled HA film by a wet process for further applications. Herein, we report the synthesis of HA nanoparticles coordinated by citric acid (Cit/HA) based on the interactions between carboxylate and calcium ions to control the sizes and shapes of the hybrid nanoparticles, to improve their dispersibility in water and to eventually form uniform transparent films with nanospaces, and investigated the film formation mechanism. As compared with the well-known rod-like HA nanoparticles (size: 48 × 15 nm2), we successfully synthesized spherical and negatively charged Cit/HA nanoparticles (size: 25 × 23 nm2) to achieve highly transparent Cit/HA films using the spin-coating technique. The Cit/HA films had uniform and crack-free appearance. About the nanostructures, we found that the Cit/HA film surfaces had meso-scaled nanospaces with a diameter of 4.2 nm based on the regular arrangement of spherical nanoparticles, instead of the HA film with a nanospace diameter of 24.5 nm formed by non-uniform accumulation. Therefore, we successfully achieved the control of the nanospace sizes of the films with the nanoparticle arrangement and realized transparent nanoparticle film formation in a very simple way, which will provide more convenient bioceramic films for biomedical applications.

Mesenchymal stem cell (MSC)-derived extracellular vesicles (EVs) are promising candidates for regenerative medicine; however, the lack of scalable methods for high quantity EV production limits their application. In addition, signature EV-derived proteins shared in 3D environments and 2D surfaces, remain mostly unknown. Herein, we present a platform combining MSC microfiber culture with ultracentrifugation purification for high EV yield. Within this platform, a high quantity MSC solution (∼3 × 108 total cells) is encapsulated in a meter-long hollow hydrogel-microfiber via coaxial bioprinting technology. In this 3D core–shell microfiber environment, MSCs express higher levels of stemness markers (Oct4, Nanog, Sox2) than in 2D culture, and maintain their differentiation capacity. Moreover, this platform enriches particles by ∼1009-fold compared to conventional 2D culture, while preserving their pro-angiogenic properties. Liquid chromatography-mass spectrometry characterization results demonstrate that EVs derived from our platform and conventional 2D culturing have unique protein profiles with 3D-EVs having a greater variety of proteins (1023 vs 605), however, they also share certain proteins (536) and signature MSC-EV proteins (10). This platform, therefore, provides a new tool for EV production using microfibers in one culture dish, thereby reducing space, labor, time, and cost.

Objectives: In this study, it was aimed to investigate the regulatory effect of N-acetylcysteine (NAC), an antioxidant, on the aging-related reactive oxygen species (ROS) production and mitochondrial membrane potential (MMP) in cardiomyocytes via exosomes released from cardiomyocytes at the molecular level. Materials and Methods: The aging model was performed by incubating rat left ventricular cell line H9c2 with D-galactose (D-Gal; 50 mg/mL) for 48 hours. Exosomes released from H9c2 pretreated with NAC were incubated with aged cardiomyocytes and their effects on the enhanced ROS production and MMP depolarization with aging were compared with both normal control cells and aged cells without exosomes. ROS and MMP measurements were calculated relative to the confocal microscope using specific fluorescent dyes (DCFDA and FCCP) by visualizing the change in fluorescence intensity. MiR-21 expression, which is also accepted as a ROS marker, and cDNA synthesis qRT-PCR measurements were performed following exosomal miRNA isolation. Results: Seventy-two-hour incubation of aging-modeled cardiomyocytes with exosomes isolated from H9c2 incubated with NAC significantly suppresses the increased ROS production associated with aging and normalizes depolarized MMP. On the other hand, NAC treatment significantly suppressed exosomal miR-21 expression in normal healthy cardiomyocytes. Conclusion: These findings indicate that ROS-targeted agents used in the treatment of heart damage may specifically suppress mitochondrionderived ROS production, which increases uncontrollably through an exosome-based paracrine mechanism. Our study also demonstrates, for the first time in the literature, that NAC induces exosomal miRNA modulation in pretreated cardiomyocytes. These findings indicate that the administration of exosomes obtained from cardiomyocytes with enhanced antioxidant system is a new treatment approach for aging-related inadequate heart function.

We recently reported that a highly homogeneous aqueous suspension of fibroin nanofiber (FNF) can be simply obtained by mechanical water-grinding a heterogeneous aqueous fibroin slurry and that the FNF in the suspension preserves the native β-sheet secondary structure during this mechanical treatment. The current study reports the surface properties of well-preserved crystalline structure novel FNF film from water-grinding preparation as compared with those of typical, conventionally prepared regenerated fibroin (RF) film. RF film was not treated with alcoholic solutions and was verified to be amorphous from a WAXD diffraction diagram. The air-side surfaces of the FNF semi-crystalline and RF amorphous films were studied to clarify differences using scanning electron microscopy (SEM), atomic force microscopy (AFM), attenuated total reflection Fourier transform infrared spectroscopy (ATR-FTIR), static water contact angle, and X-ray photoelectron spectroscopy (XPS). The well-preserved crystalline in the FNF film was found to exist near a slightly deep surface region and to act as a physically cross-linking domain, governing the molecular motions of the amorphous polypeptide chains at the very shallow surface region.

The treatment of central nervous system (CNS) pathologies is severely hampered by the presence of tightly regulated CNS barriers that restrict drug delivery to the brain. An increasing amount of data suggests that extracellular vesicles (EVs), i.e., membrane derived vesicles that inherently protect and transfer biological cargoes between cells, naturally cross the CNS barriers. Moreover, EVs can be engineered with targeting ligands to obtain enriched tissue targeting and delivery capacities. In this review, we provide a detailed overview of the literature describing a natural and engineered CNS targeting and therapeutic efficiency of different cell type derived EVs. Hereby, we specifically focus on peripheral administration routes in a broad range of CNS diseases. Furthermore, we underline the potential of research aimed at elucidating the vesicular transport mechanisms across the different CNS barriers. Finally, we elaborate on the practical considerations towards the application of EVs as a brain drug delivery system.

Skeletal muscle growth and maintenance depend on two tightly regulated processes, myogenesis and muscle regeneration. Both processes involve a series of crucial regulatory molecules including muscle-specific microRNAs, known as myomiRs. We recently showed that four myomiRs, miR-1, miR-133a, miR-133b, and miR-206, are encapsulated within muscle-derived exosomes and participate in local skeletal muscle communication. Although these four myomiRs have been extensively studied for their function in muscles, no information exists regarding their endogenous and exosomal levels across age. Here we aimed to identify any age-related changes in the endogenous and muscle-derived exosomal myomiR levels during acute skeletal muscle growth. The four endogenous and muscle-derived myomiRs were investigated in five skeletal muscles (extensor digitorum longus, soleus, tibialis anterior, gastrocnemius, and quadriceps) of 2-week-1-year-old wild-type male mice. The expression of miR-1, miR-133a, and miR-133b was found to increase rapidly until adolescence in all skeletal muscles, whereas during adulthood it remained relatively stable. By contrast, endogenous miR-206 levels were observed to decrease with age in all muscles, except for soleus. Differential expression of the four myomiRs is also inversely reflected on the production of two protein targets; serum response factor and connexin 43. Muscle-derived exosomal miR-1, miR-133a, and miR-133b levels were found to increase until the early adolescence, before reaching a plateau phase. Soleus was found to be the only skeletal muscle to release exosomes enriched in miR-206. In this study, we showed for the first time an in-depth longitudinal analysis of the endogenous and exosomal levels of the four myomiRs during skeletal muscle development. We showed that the endogenous expression and extracellular secretion of the four myomiRs are associated to the function and size of skeletal muscles as the mice age. Overall, our findings provide new insights for the myomiRs' significant role in the first year of life in mice.