Quantification of Small Extracellular Vesicles by Size Exclusion Chromatography with Fluorescence Detection

Chemical analysis of small extracellular vesicles (sEVs) circulating in body fluids holds potentials in noninvasive diagnosis of diseases and evaluation of therapeutic treatments. However, quantification of sEVs remains a challenge due to lacking of cost-effective analytical protocols. Herein we report a facile method based on size exclusion chromatography with fluorescence detection (SEC-FD) for sEVs quantification. After removal of cells and cell debris, a 0.50 mL sample (e.g., cell culture medium) is incubated with CM-Dil dye to fluorescently label sEVs. The incubation solution is then separated on a SEC column packed with Sepharose CL-4B. The eluent is monitored fluorescently at Ex553 nm/Em570 nm by using a fluorometer equipped with a 50-μL flow through cuvette. Separation efficiency of the proposed SEC-FD method was evaluated by analyzing 100 nm liposomes and albumin-FITC conjugate. Liposomes were eluted out in less than 6 min, about 10 min before albumin-FITC. A separation repeatability (RSD in retention time) of 1.4% (<i>n</i> = 5) was obtained for liposomes. In analysis of cell culture media, linear calibration curves based on SEC-FD peak height versus sEVs concentration were obtained with <i>r</i>² value of 0.996. Intraday quantification repeatability (RSD in peak height) was 3.2% (<i>n</i> = 5). The detection limit was estimated to be 2.9 × 10⁷ exosome particles/mL. The proposed assay was applied to the first study of sEVs secretion from TK6 cells cultured in serum-free medium for a culturing period from 1 to 48 h

The Combined Effect of Encapsulating Curcumin and C6 Ceramide in Liposomal Nanoparticles against Osteosarcoma

This study examines the antitumor potential of curcumin and C6 ceramide (C6) against osteosarcoma (OS) cell lines when both are encapsulated in the bilayer of liposomal nanoparticles. Three liposomal formulations were prepared: curcumin liposomes, C6 liposomes and C6-curcumin liposomes. Curcumin in combination with C6 showed 1.5 times enhanced cytotoxic effect in the case of MG-63 and KHOS OS cell lines, in comparison with curcumin liposomes alone. Importantly, C6-curcumin liposomes were found to be less toxic on untransformed primary human cells (human mesenchymal stem cells) in comparison to OS cell lines. In addition, cell cycle assays on a KHOS cell line after treatment revealed that curcumin only liposomes induced G₂/M arrest by upregulation of cyclin B1, while C6 only liposomes induced G₁ arrest by downregulation of cyclin D1. C6-curcumin liposomes induced G₂/M arrest and showed a combined effect in the expression levels of cyclin D1 and cyclin B1. The efficiency of the preparations was tested <i>in vivo</i> using a human osteosarcoma xenograft assay. Using pegylated liposomes to increase the plasma half-life and tagging with folate (FA) for targeted delivery <i>in vivo</i>, a significant reduction in tumor size was observed with C6-curcumin-FA liposomes. The encapsulation of two water insoluble drugs, curcumin and C6, in the lipid bilayer of liposomes enhances the cytotoxic effect and validates the potential of combined drug therapy

Apoptosis analysis of OS cells incubated with conditioned media from serum-deprived MSCs.

<p><b>(A and B)</b> DNA quantification of KRSOS and KHOS osteosarcoma cells treated with 0.1 μM doxorubicin for 24 h in the presence of complete culture media (control), SD-MSC media (CM) or EVs from SD-MSCs (EVs). <b>(C)</b> OS cells treated with doxorubicin in the presence of either SDM, CCM. Caspase activity measured as an increase in the amount of fluorogenic DEVD₂, a caspase 3 substrate. The data presented as the means ± SD of 3 independent experiments, *P < 0.05. <b>(D)</b> Western blot of cleaved caspase-3 in KRSOS and KHOS treatment with doxorubicin in the presence of SD-MSC EVs.</p

Effects of EVs and conditioned media from SD-MSCs on OS wound healing.

<p><b>(A)</b> KHOS cell monolayer was scratched with a p200 micropipette tip, incubated with CCM or SD-MSC conditioned media, and imaged every 6 hrs, after replacing the media with serum free media, SD-MSC conditioned media or EV free media. <b>(B)</b> Scratched KHOS cell monolayer treated with EVs from SD-MSCs for either 4 days or 12 days and imaged every 6 hrs after replacing the media with SDM + EVs. Wound closure area was determined by ImageJ software analysis. Data presented as the means of three independent measurements. * P< 0.05, ** P< 0.01, *** P< 0.005 compared to untreated controls.</p

miRNA transferred by EVs regulate PTK2 and expression.

<p><b>(A)</b> Quantitative RT-PCR showing changes in expression of four miRNA after MSC-EVs treatment. <b>(B)</b> Quantitative RT-PCR expression of downstream target PTK2/FAK. <b>(C)</b> Quantitative RT-PCR showing downregulation of FAK1repressor LKB1.</p

Comparative analysis of OS survival in the presence of mesenchymal stem cells (MSCs) or serum-deprived MSCs (SD-MSCs).

<p><b>(A)</b> KRSOS cells grown with either complete culture media (CCM) or media without serum (SDM) in the presence of transwell inserts with donor-matched MSCs or SD-MSCs. <b>(B)</b> KHOS grown in either CCM or serum-free media in the presence of inserts containing either MSC or serum-deprived MSC cells. Data presented as means ± SD, Columns, mean of three independent experiments; bars, standard deviation (SD).</p