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Background: The use of mobilizing agents for hematopoietic stem cell (HSC) transplantation is insufficient for an increasing number of patients. We previously reported lipid made endocannabinoid (eCB) ligands act on the human bone marrow (hBM) HSC migration in vitro, lacking long term stability to be therapeutic candidate. In this study, we hypothesized if a novel 2-AG-loaded polycaprolactone (PCL)-based nanoparticle delivery system that actively targets BM via phosphatidylserine (Ps) can be generated and validated. Methods: PCL nanoparticles were prepared by using the emulsion evaporation method and characterized by Zetasizer and scanning electron microscopy (SEM). The encapsulation efficiency and release profile of 2-AG were determined by liquid chromatography-tandem mass spectrometry (LC-MS/MS). The presence of cannabinoid receptors (CBRs) in HSCs and monocytes was detected by flow cytometry. Cell morphology and viability were assessed using transmission electron microscopy (TEM), SEM, and the WST-1 viability assay. The migration efficacy of the 2-AG and 2-AG-loaded nanoparticle delivery system on HSCs and HPSCs (TF-1a and TF-1) and monocytes (THP-1) was evaluated using a transwell migration assay. Results: The 140–225 nm PCL nanoparticles exhibited an increasing polydispersity index (PDI) after the addition of Ps and 2-AG, with a surface charge ranging from − 25 to -50 mV. The nanoparticles released up to 36% of 2-AG within the first 8 h. The 2-AG-Ps-PCL did not affect cellular viability compared to control on days 5 and 10. The HSCs and monocytes expressed CB1R and CB2R and revealed increased migration to media containing 1 µM 2-AG-Ps-PCL compared to control. The migration rate of the HSCs toward monocytes incubated with 1 µM 2-AG-Ps-PCL was higher than that of the monocytes of control. The 2-AG-Ps-PCL formulation provided a real time mobilization efficacy at 1 µM dose and 8 h time window via a specific CBR agonism. Conclusion: The newly generated and validated 2-AG-loaded PCL nanoparticle delivery system can serve as a stable, long lasting, targeted mobilization agent for HSCs and as a candidate therapeutic to be included in HSC transplantation (HSCT) protocols following scale-up in vivo preclinical and subsequent clinical trials. [ABSTRACT FROM AUTHOR]

The aim of this study was to analyze the existence of miRNAs derived from serum extracellular vesicles (EVs) in familial Mediterranean fever (FMF) patients. Our group has previously shown the association of certain miRNAs with FMF. Serum samples of adult and pediatric FMF patients and their age matched controls were used in the study. Serum EVs were characterized by transmission electron microscopy (TEM) and flow cytometry. RNAs were isolated from EVs and levels of miR-197-3p and miR-20a-5p were analyzed by quantitative real-time polymerase chain reaction (qRT-PCR). EV characterization using TEM demonstrated fraction of 30–120 nm-sized particles with cup-shaped morphology. Flow cytometry results revealed the CD63 and CD81 positive populations as 53.3% in serum EVs. We showed that miR-197-3p and miR-20a-5p were "circulating miRNAs" and carried in EVs of FMF patients and controls. In FMF patients, level of miR-197-3p was significantly decreased. There was no significant alteration in the level for miR-20a-5p between patients and controls. We showed the differential level of miR-197-3p in serum EVs of the FMF patients. miR-197-3p's potential as a biomarker and therapeutic target in FMF pathogenesis warrants further investigation. EVs and EV-miRNAs can be identified in FMF patients' sera. Serum EV-miR-197-3p is dysregulated in FMF patients. Serum EV-miR-197-3p might have both diagnostic and therapeutic potentials. [ABSTRACT FROM AUTHOR]

Curable approaches for primary osteosarcoma are inadequate and urge investigation of novel therapeutic formulations. Cannabinoid ligands exert antiproliferative and apoptotic effect on osteosarcoma cells via cannabinoid 2 (CB2) or transient receptor potential vanilloid type (TRPV1) receptors. In this study, we confirmed CB2 receptor expression in MG63 and Saos-2 osteosarcoma cells by qRT-PCR and flow cytometry (FCM), then reported the reduction effect of synthetic specific CB2 receptor agonist CB65 on the proliferation of osteosarcoma cells by WST-1 (water-soluble tetrazolium-1) and RTCA (real-time impedance-based proliferation). CB65 revealed an IC50 (inhibitory concentration) for MG63 and Saos-2 cells as 1.11 × 10−11 and 4.95 × 10−11 M, respectively. The specific antiproliferative effect of CB65 on osteosarcoma cells was inhibited by CB2 antagonist AM630. CB65 induced late apoptosis of MG63 and Saos-2 cells at 24 and 48 h, respectively by FCM when applied submaximal concentration. A novel CB65 liposomal system was generated by a thin film hydration method with optimal particle size (141.7 ± 0.6 nm), polydispersity index (0.451 ± 0.026), and zeta potential (−10.9 ± 0.3 mV) values. The encapsulation efficiency (EE%) of the CB65-loaded liposomal formulation was 51.12%. The CB65 and CB65-loaded liposomal formulation releasing IC50 of CB65 reduced proliferation by RTCA and invasion by scratch assay and induced late apoptosis of MG63 and Saos-2 cells, by FCM. Our results demonstrate the CB2 receptor-mediated antiproliferative and apoptotic effect of a new liposomal CB65 delivery system on osteosarcoma cells that can be used as a targeted and intelligent tool for bone tumors to ameliorate pediatric bone cancers following in vivo validation. [ABSTRACT FROM AUTHOR]

AbstractOsteoarthritis (OA) is a prevalent joint disorder characterized by cartilage and bone degradation. Medical therapies like glucosaminoglycan (GAG), chondroitin sulfate (CS), and hyaluronic acid (HA) aim to preserve joint function and reduce inflammation but may cause side effects when administered orally or via injection. Microneedle arrays (MNAs) offer a localized drug delivery method that reduces side effects. Thus, this study aims to demonstrate the feasibility of delivering GAG, CS, and HA using microneedles in vitro. An optimal needle geometry is crucial for the successful application of MNA. To address this, here we employ a multi-objective optimization framework using the non-dominated sorting genetic algorithm II (NSGA-II) to determine the ideal MNA design, focusing on preventing needle failure. Then, a three-step fabrication approach is followed to fabricate the MNAs. First, the master (male) molds are fabricated from poly(methyl methacrylate) using mechanical micromachining based on optimized needle geometry. Second, a micro-molding with Polydimethylsiloxane (PDMS) is used for the fabrication of production (female) molds. In the last step, the MNAs were fabricated by microcasting the hydrogels using the production molds. Light microscopy (LIMI) confirms the accuracy of the MNAs manufactured, and in vitro skin insertion tests demonstrate failure-free needle insertion. Subsequently, we confirmed the biocompatibility of MNAs by evaluating their impact on the L929 fibroblast cell line, human chondrocytes, and osteoblasts. [ABSTRACT FROM AUTHOR]

Flow cytometry and immunohistochemistry techniques both determine the target protein by immunolabeling. Flow cytometric analysis quantifies total number of fluorescent labeled cells and qualify sup-populations according to size and granularity. Immunohistochemistry is able to map immune-labeled cells and extracellular matrix components under light and electron microscope by enzyme or fluorescent molecules. Real-time identification, in-time classification, and final plotting of spermatogonial lineage are of crucial importance for monitoring the fertility potential of spermatogonial stem cell microenvironment and predicting progress of spermatogenesis. Here we define the evaluation of mouse male germ cell microenvironment at single cell and whole tissue section levels by using flow cytometric and immunohistochemical approaches., (© 2023. Springer Science+Business Media, LLC.)