A Perspective on the Characterization of Early Neural Progenitor Cell-Derived Extracellular Vesicles for Targeted Delivery to Neuroblastoma Cells.

As an element of the cellular signaling systems, extracellular vesicles (EVs) exhibit many desirable traits for usage as targeted delivery vehicles. When administered, EVs cause little to no toxic or immune response, stay in circulation for longer periods compared to synthetic carriers, preferentially accumulate in tissues that are the same or similar to their cell-of-origin and can pass through the blood-brain barrier. Combined, these traits make neural EVs a particularly promising tool for delivering drugs to the brain. This study aims to combine tissue and EVs engineering to prepare neural differentiated cells derived EVs that exhibit neural properties, to develop an effective, tissue-homing drug and gene delivery platform for the brain. Early neural differentiated cell-derived EVs were produced with neural characteristics from neural differentiated human neonatal dermal fibroblasts. The EVs carried key neural proteins such as Nestin, Sox2 and Doublecortin. The cellular uptake of early neural differentiated cell-derived EVs was higher compared to non-neural EVs during in vitro uptake assays on neuroblastoma cells. Moreover, eND-EVs were significantly decreased the viability of neuroblastoma cells. In conclusion, this study revealed that early neural differentiated cell-derived EVs have potential as a promising drug carrier for the treatment of various neural disorders.

Extracellular vesicles derived from human foreskin cells (hFS-Exo) accelerate cell migration and angiogenesis through MAPK pathway: an in vitro study.

BACKGROUND: Wound healing is one of the important processes in the body. Attempts to create new drugs are of interest due to the side effects of natural and chemical wound healing compounds. To overcome this obstacle, stem cells have been used as healing agents. However, both difficulties in collection and risks such as rejection and teratoma in the recipient body have limited the use of stem cells, directly. Since the potential content of the stem cells can be transferred to the recipient cells by vesicles, small extracellular vesicles have recently become prominent agents. METHODS AND RESULTS: The wound-healing effect of extracellular vesicles derived from foreskin cells was investigated in both keratinocyte and endothelial cells. Migration assay, RT-PCR, Col1a1 ELISA and Western Blot experiments were utilized to reveal healing effect of EVs and its possible molecular pathways. EV-treated groups exhibited more proliferative, invasive, and migrative characteristics. When comparing to the control group, new vessel formation was induced in EV groups. An increase in gene levels of growth factors related to wound healing and change in the mitogen-activated protein kinase (MAPK) signaling pathway proteins in EV-treated groups were determined. Possible molecular mechanisms underlying cell movements were associated with the MAPK pathway. It was found that human foreskin cell EVs (hFS-Exo) may have a potential to heal wounds in a short period of time by triggering the MAPK pathway. CONCLUSIONS: hFS-Exo could be a new promising wound healing agent in the future.

Immunomodulatory Behavior of Mesenchymal Stem Cells.

The use of Mesenchymal Stem Cells (MSCs) in the treatment of diseases where immunomodulation impacts therapy is increasing steadily. Recent studies aim to achieve effective use of MSCs in treatment of Graft versus Host Disease (GvHD), Crohn's disease and organ transplantations. The molecular mechanisms governing immunomodulatory properties of MSCs have not been fully understood, although current studies are indicating progress. Especially, in vitro studies and animal models provide a major contribution to our knowledge in clinical use of MSCs. The immunosuppressive and immune-enhancer properties of MSCs are -typically- determined with respect to type and concentrations of soluble molecules found in their physiological environment. In mammals the immune system protects the organism -not only- from certain microorganisms, but also from any entity that it recognizes as foreign, including its own cells when it is received as a threat. This protection can sometimes occur by increasing the number of immune cells and sometimes by suppressing a pathologically hyper-induced immunological response. In particular, realization of the bi-directional effect of MSCs on immune cells has placed substantial emphasis on this area of research. This chapter focuses on the interaction of MSCs with the immune cells, the bilateral role of these interactions, and whether studies that aim to understand these interactions can yield promising results in terms of developing improved use of MSCs in treatment.

Unveiling the potential: Extracellular vesicles from plant cell suspension cultures as a promising source.

Extracellular vesicles are secreted by all eukaryotic cells and they have an important role in intercellular signaling. Plant extracellular vesicles (PEVs) are a novel area of research that has gained attention due to their potential implications in biomolecule transport and therapeutic applications. PEVs are lipid bilayer-enclosed structures that contain a diverse cargo of biomolecules such as proteins and lipids. Moreover, it is known that PEVs have a noticeable therapeutic potential for various conditions such as inflammation and oxidative stress. However, there are critical problems such as removing the endosomes and plant-derived biomolecules that decrease the standardization and therapeutic efficacy of PEVs. In our study, the aim was to characterize plant cell suspension-derived extracellular vesicles (PCSEVs) obtained from two different plant cell suspension cultures: Stevia rebaudiana and Vaccaria hispanica. These vesicles were isolated using ultrafiltration and characterized with nanoparticle tracking analysis (NTA) and atomic force microscopy (AFM). The molecular composition of PCSEVs was profiled and the cellular uptake assay was performed. Our results demonstrated that PCSEVs have a spherical shape, less than 200 nm. In the fatty acid analysis, the primary components in PCSEVs were palmitic acid, linoleic acid, and cis-vaccenic acid. The protein content of Stevia rebaudiana-derived EVs (SDEVs) was largely associated with proteins involved in extracellular structures and functions. Conversely, Vaccaria hispanica-derived EVs (HDEVs) displayed a higher presence of cytosolic proteins. These findings contribute to the understanding of PCSEVs and open up potential avenues in extracellular vesicle research, pointing to promising prospects for future innovations in various fields.

The emerging role of breast cancer derived extracellular vesicles-mediated intercellular communication in ovarian cancer progression and metastasis.

Breast cancer is one of the most occurring cancer types in women worldwide and metastasizes to several organs such as bone, lungs, liver, brain, and ovaries. Extracellular vesicles (EVs) mediate intercellular signaling which has a profound effect on tumor development and metastasis. Recent developments in the field of EVs provide an opportunity to investigate the roles of EVs released from tumor cells in metastasis. In this study, we compared the effects of metastatic breast cancer-derived EVs on both nonluteinized granulosa HGrC1 and ovarian cancer OVCAR-3 cells in terms of proliferation, invasion, apoptosis, and gene expression levels. EVs were isolated from the culture medium of metastatic breast cancer cell line MDA-MB-231 by ultracentrifugation. Cell proliferation, apoptosis, cell cycle, invasion, and cellular uptake analysis were performed to clarify the roles of tumor-derived EVs in both cells. 6.85 × 108 nanoparticles of BCD-EVs were markedly increased cell proliferation as well as invasion capacity. Exposing the cells with BCD-EVs for 24 h, resulted in an accumulation of both cells in G2/M phase as determined by flow cytometry. The apoptosis assay results were consistent with cell proliferation and cell cycle results. The uptake of the BCD-EVs was efficiently internalized by both cells. In addition, marked variations in fatty acid composition between cells were observed. BCD-EVs appeared new fatty acids in HGrC1. Besides, BCD-EVs upregulated epithelial-mesenchymal transition (EMT) and proliferation-related genes. In conclusion, an environment of tumor-derived EVs changes the cellular phenotype of cancer and noncancerous cells and may lead to tumor progression and metastasis.

Endothelial cell-derived extracellular vesicles induce pro-angiogenic responses in mesenchymal stem cells.

Angiogenesis is a central component of vital biological processes such as wound healing, tissue nourishment, and development. Therefore, angiogenic activities are precisely maintained with secreted factors such as angiopoietin-1 (Ang1), fibroblast growth factor (FGF), and vascular endothelial growth factor (VEGF). As an element of intracellular communication, extracellular vesicles (EVs)-particularly EVs of vascular origin-could have key functions in maintaining angiogenesis. However, the functions of EVs in the control of angiogenesis have not been fully studied. In this study, human umbilical vein endothelial cell line (HUVEC)-derived small EVs (<200 nm; HU-sEVs) were investigated as a potential pro-angiogenic agent. Treating mesenchymal stem cells (MSCs) and mature HUVEC cells with HU-sEVs induced their tube formation under in vitro conditions and significantly increased the expression of angiogenesis-related genes, such as Ang1, VEGF, Flk-1 (VEGF receptor 2), Flt-1 (VEGF receptor 1), and vWF (von Willebrand Factor), in a dose-dependent manner. These results indicate that HU-sEVs take part in angiogenesis activities in physiological systems, and suggest endothelial EVs as a potential therapeutic candidate for the treatment of angiogenesis-related diseases.

Lead Borate Nanoparticles Induce Apoptotic Gene Activity in P53 Mutant Cancer Cells.

Cancer is a complex and multistage disease that causes suffering worldwide. Several mutations in tumor suppressor proteins are mostly responsible for tumorigenic development. Thus, determination of the mutations and developing a mutation targeted therapy are crucial in order to cure cancer. Moreover, since healthy cells do not have mutations in their tumor suppressor genes, mutation-specific treatment is responsible for selective treatment without harming a healthy tissue in the body. In this current study, lead borate nanoparticles (LB-Np) have been synthesized, and their effects on P53 mutant cancer cells were investigated. The synthesis method includes steps of mixing a borate buffer solution with the lead nitrate solution, washing the resulting precipitate with distilled water and eventually preparing stable LB-Np solutions. Cell viability analysis was conducted to identify the toxicity of LB-Np in HaCaT, A549, MCF7, and T47D cell lines. The changes in morphologies of breast cancer cell lines were demonstrated by using microscopical analysis. Additionally, alterations in gene expressions were determined in breast cancer cell lines after LB-Np treatment. This multidisciplinary study also identified the selective effect of LB-Np in cancer cell lines, in vitro. MTS and quantitative polymerase chain reaction assays demonstrated the effect of LB-Np were specific for p53 mutation cell line, T47D. Breast cancer cell line T47D has 580 C/T mutation which affects the activation of p53 tumor suppressor protein. However, LB-Np treatment effectively killed T47D cell lines and did not affect any other cell lines that have no p53 mutations such as MCF7, A549, and healthy HaCaT. Overall, synthesized LB-Np were found to be effective in p53-mutated cell lines and showed a remarkable selective anti-cancer activity.

A pilot study for treatment of severe COVID-19 pneumonia by aerosolized formulation of convalescent human immune plasma exosomes (ChipEXO™).

This is a single-center prospective, open-label, single arm interventional study to test the safety and efficacy of recently described ChipEXO™ for severe COVID-19 pneumonia. The ChipEXO™ is a natural product derived from convalescent human immune plasma of patients recovered from moderate COVID-19 infection. In September 2021, 13 patients with pending respiratory failure were treated with ChipEXO™ adapted for aerosolized formulation delivered via jet nebulizer. Patients received 1-5x1010 nano vesicle/5 mL in distilled water twice daily for five days as an add-on to ongoing conventional COVID-19 treatment. The primary endpoint was patient safety and survival over a 28-day follow-up. The secondary endpoint was longitudinal assessment of clinical parameters following ChipEXO™ to evaluate treatment response and gain insights into the pharmacodynamics. ChipEXO™ was tolerated well without any allergic reaction or acute toxicity. The survival rate was 84.6% and 11 out of 13 recovered without any sequel to lungs or other organs. ChipEXO™ treatment was effective immediately as shown in arterial blood gas analyses before and two hours after exosome inhalation. During the 5 days of treatment, there was a sustainable and gradual improvement on oxygenation parameters: i.e. respiratory rate (RR) [20.8% (P < 0.05)], oxygen saturation (SpO2) [6,7% (P < 0.05)] and partial pressure of oxygen to the fraction of inspired oxygen (PaO2/FiO2) [127.9% (P < 0.05)] that correlated with steep decrease in the disease activity scores and inflammatory markers, i.e. the sequential organ failure assessment (SOFA) score (75%, p < 0.05), C-reactive protein (46% p < 0.05), ferritin (58% p = 0.53), D-dimer (28% p=0.46). In conclusion, aerosolized ChipEXO™ showed promising safety and efficacy for life-threatening COVID-19 pneumonia. Further studies on larger patient populations are required to confirm our findings and understand the pathophysiology of improvement toward a new therapeutic agent for the treatment of severe COVID-19 pneumonia.

Preclinical Studies on Convalescent Human Immune Plasma-Derived Exosome: Omics and Antiviral Properties to SARS-CoV-2.

The scale of the COVID-19 pandemic forced urgent measures for the development of new therapeutics. One of these strategies is the use of convalescent plasma (CP) as a conventional source for passive immunity. Recently, there has been interest in CP-derived exosomes. In this report, we present a structural, biochemical, and biological characterization of our proprietary product, convalescent human immune plasma-derived exosome (ChipEXO), following the guidelines set forth by the Turkish Ministry of Health and the Turkish Red Crescent, the Good Manufacturing Practice, the International Society for Extracellular Vesicles, and the Gene Ontology Consortium. The data support the safety and efficacy of this product against SARS-CoV-2 infections in preclinical models.

Effective Scarless Wound Healing Mediated by Erbium Borate Nanoparticles.

The developments of nanoparticle-based treatments that benefit from novel discoveries have an essential place in the regeneration of acute and chronic wounds. Furthermore, research about the treatment methods which attempt to swiftly and scarless wound recovery has increased over time. In recent years, it has been shown that metallic-based nanoparticles, especially silver and gold derived, have an accelerating effect on chronic and contaminated wound healing. The crucial factors of inducing and completion of regeneration of wound are enhanced epithelialization rate and neovascularization in the tissue. In our study, the main purpose is the investigation of the boosting effects of erbium borate nanoparticles on the wound healing process, especially scarless ones. Newly syntesized erbium borate nanoparticles (ErB-Nps) were characterized by their concentration and particle size using nanoparticle tracking analysis (NTA). In order to examine the effect of ErB-Np on wound closure, scratch assay for dermal epithelial cells and tube formation assay for endothelial cells were performed. In addition, in order to examine the effect of the ErB-Np at a molecular level, the levels of genes related to both wound healing, inflammation, and scarless wound closure were determined with the RT-PCR experiment. Consequently, it has been shown that erbium borate nanoparticles have increased the melioration speed of scar tissue and have given clues about scarless healing potential. The investigation of the regeneration potential of erbium borate nanoparticles was done via MTS assay, quantitative PCR analysis, reactive oxygen species assay, and scratch assay. Our results show that ErB-Np is a proper agent that can be used for scarless wound healing.

Sodium Pentaborate Pentahydrate ameliorates lipid accumulation and pathological damage caused by high fat diet induced obesity in BALB/c mice.

BACKGROUND: Obesity is one of the most popular topic in the field of research. In order to defeat this highly widespread disease, the mechanism of fat accumulation at the molecular level and its elimination are crucial. The use of boron has been showing promising results during the recent years. METHODS: In this study, anti-obesity potential of Sodium Pentaborate Pentahydrate (SPP) used as a dietary supplement on BALB/c mice fed with a high-fat diet was evaluated. Mice were divided into four groups with different diets, consisting of a normal diet, a high-fat diet (HFD) (containing 60 % fat), a HFD-supplemented with 0.5 mg/g body weight (BW) of SPP and a HFD-supplemented with 1.5 mg/g body weight (BW) of SPP. The animals were then observed for 10 weeks and physically monitored, and were sacrificed at the end of the experiment for physical and physicochemical evaluation. RESULTS: According to the physical parameters measured -body weight, food and water intake ratios-, the results indicate that SPP decreased weight gain in a dose dependent manner. Measurement of the hormone levels in the blood and fat accumulation in organs of mice also supported the anti-obesity effects of SPP. Expressions of adipogenesis related genes were also negatively regulated by SPP administration in white adipose tissue (WAT) tissue. CONCLUSION: These findings promise a treatment approach and drug development that can be used against obesity when SPP is used in the right doses. As a future aspect, clinical studies with SPP will reveal the effect of boron derivatives on obesity.

Grapefruit-derived extracellular vesicles as a promising cell-free therapeutic tool for wound healing.

Due to the prevalence of individuals suffering from chronic wounds, developing safe and effective wound care agents are one of the more prominent fields of research in biology. However, wound healing is a complex, multi-stage biological process, involving multiple sequences of biological responses from different types of cells, secreted mediators, and extracellular matrix elements. Plants have a long history of use in the treatment of wounds. Plant-derived extracellular vesicles, which are secreted nano vesicle messengers responsible for intercellular communications, show promise as a new, biotechnological wound-care agent. In this study, we assessed the wound healing potential of extracellular vesicles isolated from grapefruits - a plant with well-known anti-inflammatory and wound healing properties. Grapefruit extracellular vesicles (GEVs) increased cell viability and cell migration while reducing intracellular ROS production in a dose-dependent manner in HaCaT cells. Expression of proliferation and migration-related genes were raised by GEV treatment in a dose dependent manner. Additionally, GEV treatment increased the tube formation capabilities of treated HUVEC cells. These findings suggest that GEVs can be used as plant-derived wound healing agents, and have shown potential as a biotechnological agent for wound healing. Further development and study of plant-derived extracellular vesicles may lead to the realization of their full potential.

Optimized Isolation of Extracellular Vesicles From Various Organic Sources Using Aqueous Two-Phase System.

From biomarkers to drug carriers, Extracellular Vesicles (EVs) are being used successfully in numerous applications. However, while the subject has been steadily rising in popularity, current methods of isolating EVs are lagging behind, incapable of isolating EVs at a high enough quantity or quality while also requiring expensive, specialized equipment. The "isolation problem" is one of the major obstacles in the field of EV research - and even more so for their potential, widespread use for clinical diagnosis and therapeutic applications. Aqueous Two-Phase Systems (ATPS) has been reported previously as a promising method for isolating EVs quickly and efficiently, and with little contaminants - however, this method has not seen widespread use. In this study, an ATPS-based isolation protocol is used to isolate small EVs from plant, cell culture, and parasite culture sources. Isolated EVs were characterized in surface markers, size, and morphological manner. Additionally, the capacity of ATPS-based EV isolation in removing different contaminants was shown by measuring protein, fatty acid, acid, and phenol red levels of the final isolate. In conclusion, we have shown that EVs originating from different biological sources can be isolated successfully in a cost-effective and user-friendly manner with the use of aqueous two-phase systems.