Extracellular vesicle-based formulation of doxorubicin: drug loading optimization, characterization, and cytotoxicity evaluation in tumor spheroids.

Doxorubicin (DOX) is a chemotherapeutic with considerable efficacy, but its application is limited due to cardiotoxicity. Nanoparticles can improve DOX efficacy and prevent its adverse effects. Herein, DOX-loaded extracellular vesicles (DOX-EVs) were prepared using different loading methods including incubation, electroporation, and sonication in different hydration buffers to permeabilize nanostructures or desalinize DOX for improved entrapment. Different protein:drug (µg:µg) ratios of 1:10, 1:5, and 1:2, and incubation parameters were also investigated. The optimal formulation was characterized by western blotting, electron microscopy, Zetasizer, infrared spectroscopy, and release study. The cellular uptake and efficacy were investigated in MCF-7 spheroids via MTS assay, spheroid formation assay (SFA), confocal microscopy, and flow cytometry. The percentage of entrapment efficiency (EE) of formulations was improved from 1.0 ± 0.1 to 22.0 ± 1.4 using a protein:drug ratio of 1:2 and sonication in Tween 80 (0.1%w/v) containing buffer. Characterization studies verified the vesicles' identity, spherical morphology, and controlled drug release properties. Cellular studies revealed the accumulation and cytotoxicity of DOX-EVs in the spheroids, and SFA and confocal microscopy confirmed the efficacy and cellular localization. Flow cytometry results revealed a comparable and amplified efficacy for DOX-EV formulations with different cell origins. Overall, the EV formulation of DOX can be applied as a promising alternative with potential advantages.

Characterisation of extracellular vesicles isolated from hydatid cyst fluid and evaluation of immunomodulatory effects on human monocytes.

Hydatidosis is a disease caused by the larval stage of Echinococcus granulosus, which involves several organs of intermediate hosts. Evidence suggests a communication between hydatid cyst (HC) and hosts via extracellular vesicles. However, a little is known about the communication between EVs derived from HC fluid (HCF) and host cells. In the current study, EVs were isolated using differential centrifugation from sheep HCF and characterized by western blot, electron microscope and size distribution analysis. The uptake of EVs by human monocyte cell line (THP-1) was evaluated. The effects of EVs on the expression levels of pro- and anti-inflammatory cytokines were investigated using quantitative real-time PCR (RT-PCR), 3 and 24 h after incubation. Moreover, the cytokine level of IL-10 was evaluated in supernatant of THP-1 cell line at 3 and 24 h. EVs were successfully isolated and showed spherical shape with size distribution at 130.6 nm. After 3 h, the expression levels of pro-inflammatory cytokine genes (IL1Β, IL15 and IL8) were upregulated, while after 24 h, the expression levels of pro-inflammatory cytokines were decreased and IL13 gene expression showed upregulation. A statistically significant increase was seen in the levels of IL-10 after 24 h. The main mechanism of the communication between EVs derived from HCF and their host remains unclear; however, time-dependent anti-inflammatory effects in our study suggest that HC may modulate the immune responses via EVs.

Milk-Derived Extracellular Vesicles: Biomedical Applications, Current Challenges, and Future Perspectives.

Extracellular vesicles (EVs) are nano to-micrometer-sized sacs that are released by almost all animal and plant cells and act as intercellular communicators by transferring their cargos between the source and target cells. As a safe and scalable alternative to conditioned medium-derived EVs, milk-derived EVs (miEVs) have recently gained a great deal of popularity. Numerous studies have shown that miEVs have intrinsic therapeutic actions that can treat diseases and enhance human health. Additionally, they can be used as natural drug carriers and novel classes of biomarkers. However, due to the complexity of the milk, the successful translation of miEVs from benchtop to bedside still faces several unfilled gaps, especially a lack of standardized protocols for the isolation of high-purity miEVs. In this work, by comprehensively reviewing the bovine miEVs studies, we provide an overview of current knowledge and research on miEVs while highlighting their challenges and enormous promise as a novel class of theranostics. It is hoped that this study will pave the way for clinical applications of miEVs by addressing their challenges and opportunities.

Sirolimus-loaded exosomes as a promising vascular delivery system for the prevention of post-angioplasty restenosis.

Restenosis remains the main reason for treatment failure of arterial disease. Sirolimus (SIR) as a potent anti-proliferative agent is believed to prevent the phenomenon. The application of exosomes provides an extended-release delivery platform for SIR intramural administration. Herein, SIR was loaded into fibroblast-derived exosomes isolated by ultracentrifugation. Different parameters affecting drug loading were optimized, and exosome samples were characterized regarding physicochemical, pharmaceutical, and biological properties. Cytotoxicity, scratch wound assays, and quantitative real-time PCR for inflammation- and migration-associated genes were performed. Restenosis was induced by carotid injury in a rat carotid model and then exosomes were locally administered. After 14 days, animals were investigated by computed tomography (CT) angiography, morphometric, and immunohistochemical analyses. Western blotting confirmed the presence of specific protein markers in exosomes. Characterization of empty and SIR-loaded exosomes verified round and nanoscale structure of vesicles. Among prepared formulations, desired entrapment efficiency (EE) of 76% was achieved by protein:drug proportion of 2:1 and simple incubation for 30 min at 37 °C. Also, the optimal formulation released about 30% of the drug content during the first 24 h, followed by a prolonged release for several days. In vitro studies revealed the uptake and functional efficacy of the optimized formulation. In vivo studies revealed that %restenosis was in the following order: saline > empty exosomes > SIR-loaded exosomes. Furthermore, Ki67, alpha smooth muscle actin (α-SMA), and matrix metalloproteinase (MMP) markers were less expressed in the SIR-exosomes-treated arteries. These findings confirmed that exosomal SIR could be a hopeful strategy for the prevention of restenosis.

Proteomic profiling of mesenchymal stem cell-derived extracellular vesicles: Impact of isolation methods on protein cargo.

Extracellular vesicles (EVs) are nanosized vesicles with a lipid bilayer that are secreted by cells and play a critical role in cell-to-cell communication. Despite the promising reports regarding their diagnostic and therapeutic potential, the utilization of EVs in the clinical setting is limited due to insufficient information about their cargo and a lack of standardization in isolation and analysis methods. Considering protein cargos in EVs as key contributors to their therapeutic potency, we conducted a tandem mass tag (TMT) quantitative proteomics analysis of three subpopulations of mesenchymal stem cell (MSC)-derived EVs obtained through three different isolation techniques: ultracentrifugation (UC), high-speed centrifugation (HS), and ultracentrifugation on sucrose cushion (SU). Subsequently, we checked EV marker expression, size distribution, and morphological characterization, followed by bioinformatic analysis. The bioinformatic analysis of the proteome results revealed that these subpopulations exhibit distinct molecular and functional characteristics. The choice of isolation method impacts the proteome of isolated EVs by isolating different subpopulations of EVs. Specifically, EVs isolated through the high-speed centrifugation (HS) method exhibited a higher abundance of ribosomal and mitochondrial proteins. Functional apoptosis assays comparing isolated mitochondria with EVs isolated through different methods revealed that HS-EVs, but not other EVs, induced early apoptosis in cancer cells. On the other hand, EVs isolated using the sucrose cushion (SU) and ultracentrifugation (UC) methods demonstrated a higher abundance of proteins primarily involved in the immune response, cell-cell interactions and extracellular matrix interactions. Our analyses unveil notable disparities in proteins and associated biological functions among EV subpopulations, underscoring the importance of meticulously selecting isolation methods and resultant EV subpopulations based on the intended application.

Thiolated chitosan hydrogel-embedded niosomes: A promising crocin delivery system toward the management of aphthous stomatitis.

Aphthous stomatitis is a common inflammatory oral disease with challenging management. Crocin is a natural carotenoid that has shown great anti-inflammatory properties. The aim of this study was to develop thiolated chitosan (TCS)-based hydrogels containing niosomes to serve as a mucoadhesive crocin delivery system for aphthous stomatitis. Crocin-loaded niosomes were prepared and the impact of surfactant type, cholesterol content, and lipid to drug ratio on the characteristics of niosomes was evaluated. TCS was synthesized and the success of thiolation was investigated. The optimum niosomal formulation was loaded into the hydrogel and the hybrid system was characterized regarding the morphology, mucoadhesive properties, viscosity, chemical structure, in vitro drug release, and in vivo efficacy. The optimized niosome formulation showed 77% crocin entrapment, a particle diameter of 59 nm, and a zeta potential of -18 mV. The niosome-containing hydrogel exhibited pseudoplastic rheological behavior, mucoadhesive properties, suitable swelling, and sustained release of crocin. In vivo study revealed that the niosome-containing hydrogel improved ulcer healing and decreased the expression of tumor necrosis factor-alpha (TNF-α) and p53 while increasing the expression of vascular endothelial growth factor (VEGF) and alpha-smooth muscle actin (α-SMA). Collectively, TCS hydrogel-embedded crocin-loaded niosomes is a promising therapeutic option for aphthous stomatitis. CHEMICAL COMPOUNDS STUDIED IN THIS ARTICLE: Crocin (PubChem CID: 5281233) Chitosan (PubChem CID: 71853) Thioglycolic acid (PubChem CID: 1133) 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (PubChem CID: 2723939) 5,5'-dithiobis (2-nitrobenzoic acid) (PubChem CID: 6254) Cholesterol (PubChem CID: 5997).

Exosomes as cutting-edge therapeutics in various biomedical applications: An update on engineering, delivery, and preclinical studies.

Exosomes are cell-derived nanovesicles, circulating in different body fluids, and acting as an intercellular mechanism. They can be purified from culture media of different cell types and carry an enriched content of various protein and nucleic acid molecules originating from their parental cells. It was indicated that the exosomal cargo can mediate immune responses via many signaling pathways. Over recent years, the therapeutic effects of various exosome types were broadly investigated in many preclinical studies. Herein, we present an update on recent preclinical studies on exosomes as therapeutic and/or delivery agents for various applications. The exosome origin, structural modifications, natural or loaded active ingredients, size, and research outcomes were summarized for various diseases. Overall, the present article provides an overview of the latest exosome research interests and developments to clear the way for the clinical study design and application.

Amphotericin B-Loaded Extracellular Vesicles Derived from Leishmania major Enhancing Cutaneous Leishmaniasis Treatment through In Vitro and In Vivo Studies.

Background: Recent studies have shown an increasing number of patients with cutaneous leishmaniasis (CL) who do not respond to pentavalent antimonials as the first line of treatment for CL. Nanocarriers such as extracellular vesicles (EVs) are efficient vehicles that might be used as drug delivery systems for the treatment of diseases. Therefore, we aimed to isolate and characterize the EVs of Leishmania major, load them with Amphotericin B (AmB), and investigate the toxicity and efficacy of the prepared drug form. Methods: The EVs of L. major were isolated, characterized, and loaded with amphotericin B (AmB), and the EVs-Amphotericin B (EVs-AmB) form was synthesized. Relevant in vitro and in vivo methods were performed to evaluate the toxicity and efficacy of EVs-AmB compared to the control. Results: The anti-leishmanial activity of the EVs-AmB showed a higher percentage inhibition (PI%) (P = 0.023) compared to the AmB at different concentrations and time points. Obtained data showed a significant increase in the lesion size and parasite load in the lesion, PBS, and EVs mice groups in comparison with EVs-AmB, AmB, and Glucantime groups (P < 0.05), EVs-AmB had a significant decrease in lesion sizes in comparison with AmB (P < 0.05). Results showed that EVs-AmB decreased its toxicity to the kidneys and liver (P < 0.05). Conclusion: EVs-AmB improved the efficacy of AmB in mouse skin lesions and reduced hepatorenal toxicity. Furthermore, EVs could be a promising nanoplatform for the delivery of AmB in CL caused by L. major.

Applications of Novel and Nanostructured Drug Delivery Systems for the Treatment of Oral Cavity Diseases.

PURPOSE: Novel drug delivery systems (DDSs) hold great promise for the treatment of oral cavity diseases. The main objective of this article was to provide a detailed overview regarding recent advances in the use of novel and nanostructured DDSs in alleviating and treating unpleasant conditions of the oral cavity. Strategies to maximize the benefits of these systems in the treatment of oral conditions and future directions to overcome these issues are also discussed. METHODS: Publications from the last 10 years investigating novel and nanostructured DDSs for pathologic oral conditions were browsed in a systematic search using the PubMed/MEDLINE, Web of Science, and Scopus databases. Research on applications of novel DDSs for periodontitis, oral carcinomas, oral candidiasis, xerostomia, lichen planus, aphthous stomatitis, and oral mucositis is summarized. A narrative exploratory review of the most recent literature was undertaken. FINDINGS: Conventional systemic administration of therapeutic agents could exhibit high clearance of drugs from the bloodstream and low accumulation at the target site. In contrast, conventional topical systems face problems such as short residence time in the affected region and low patient compliance. Novel and nanostructured DDSs are among the most effective and commonly used methods for overcoming the problems of conventional DDSs. The main advantages of these systems are that they possess the ability to protect active agents from systemic and local clearance, enhance bioavailability and cellular uptake, and provide immediate or modified release of therapeutic agents after administration. In the design of local drug delivery devices such as nanofiber mats, films, and patches, components and excipients can significantly affect factors such as drug release rate, residence time in the oral cavity, and taste in the mouth. Choosing appropriate additives is therefore essential. IMPLICATIONS: Local drug delivery devices such as nanofiber mats, nanoparticles, liposomes, hydrogels, films, and patches for oral conditions can significantly affect drug efficacy and safety. However, more precise clinical studies should be designed and conducted to confirm promising in vitro and in vivo results. In recent years, novel and nanostructured DDSs increasingly attracted the attention of researchers as a means of treatment and alleviation of oral diseases and unpleasant conditions. However, more clinical studies should be performed to confirm promising in vitro and in vivo results. To transform a successful laboratory model into a marketable product, the long-term stability of prepared formulations is essential. Also, proper scale-up methods with optimum preparation costs should be addressed.

Exosomes as a next-generation drug delivery system: An update on drug loading approaches, characterization, and clinical application challenges.

Exosomes are small nanoparticles secreted by almost all cells and have a well-known role in intercellular communication. They are found in different body fluids and can also be isolated from cell culture media. They contain a natural cargo including various protein and nucleic acid molecules originated from their donor cells. In recent years, exosomes have emerged as a desired drug delivery system. They are believed to provide a targeted delivery of drug molecules, supplemented with their natural function. Furthermore, they have a membranous structure similar to liposomes, and that motivated researchers to apply their previous experience of drug loading into liposomes for exosomes. Herein, we discuss applied methods for the encapsulation of different drugs into exosomes, parameters affecting the incorporation of drug molecules into exosomes, characterization techniques, recent achievements, commercialization challenges and the potential future developments of exosomal drugs. Overall, while the application of exosomes as a drug delivery system is still in its infancy, they are considered to be a new class of natural nanocarriers with great potential for clinical application. Understanding of their key formulation parameters, pharmaceutical properties, in vivo behavior and applicable scale-up production will pave their way to the market. STATEMENT OF SIGNIFICANCE: Details of loading methods, characterization and biopharmaceutical properties of drug-incorporated exosomes are presented. Most parameters affecting encapsulation of drugs into exosomes are mentioned to serve as a guide for future studies in this field. Moreover, challenges on the way of exosomes to the market and clinic are described.

Potential application of liposomal nanodevices for non-cancer diseases: an update on design, characterization and biopharmaceutical evaluation.

Liposomes, lipid-based vesicular systems, have attracted major interest as a means to improve drug delivery to various organs and tissues in the human body. Recent literature highlights the benefits of liposomes for use as drug delivery systems, including encapsulating of both hydrophobic and hydrophilic cargos, passive and active targeting, enhanced drug bioavailability and therapeutic effects, reduced systemic side effects, improved cargo penetration into the target tissue and triggered contents release. Pioneering work of liposomes researchers led to introduction of long-circulating, ligand-targeted and triggered release liposomes, as well as, liposomes containing nucleic acids and vesicles containing combination of cargos. Altogether, these findings have led to widespread application of liposomes in a plethora of areas from cancer to conditions such as cardiovascular, neurologic, respiratory, skin, autoimmune and eye disorders. There are numerous review articles on the application of liposomes in treatment of cancer, which seems the primary focus, whereas other diseases also benefit from liposome-mediated treatments. Therefore, this article provides an illustrated detailed overview of liposomal formulations, in vitro characterization and their applications in different disorders other than cancer. Challenges and future directions, which must be considered to obtain the most benefit from applications of liposomes in these disorders, are discussed.