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Stem cells derived exosomes as biological nano carriers for VCR sulfate for treating breast cancer stem cells – Scientific Reports

Isolation and culturing of bone marrow mesenchymal stem cells

A 3-week-old rat was euthanized using cervical dislocation. Its femur and tibia were isolated as stated in the protocol of18. The bone marrow was flushed from the bones into a T-25 flask containing Low glucose DMEM supplemented with 1% antibiotic and 10% FBS. The flask was incubated at 37 °C in 5% CO2 and 95% humidity. The culture medium was removed and replaced with fresh medium every 3–4 days. When the cells reached 70–80% confluence, they were subcultured after using 0.25% trypsin–EDTA for 3 min at 37 °C. 1ml of complete medium was used to inhibit the action of trypsin, and then recentrifuged 10 min at 200×g. The cell pellet was reconstituted in a complete medium and replated in a new T-25 flask19. This process was repeated four times, resulting in four passages of cells.

Identification and characterization of MSCs

Morphological analysis

When MSC culture was on its fourth passage the morphological traits of MSCs were examined under an inverted microscope.

Flow cytometric analysis

After cells were washed and resuspended, the following fluorescein isothiocyanate-conjugated monoclonal antibodies were incubated in PBS supplemented with 3% FBS: anti-CD90 (554898, BD Pharmingen), 200; anti-CD73 (550,741, BD Pharmingen), and anti-CD105 (550,546, BD Pharmingen). Samples were analyzed using a forward scattering analytical method (Becton–Dickinson, Canada)20,21.

MSCs derived exosomes isolation and characterization

For the extraction of exosomes, Passage IV of MSCs (5 × 106 cells/mL) was utilized. The MSCs were cultured in RPMI medium without FBS and treated with 0.5% BSA (Sigma). The cell-free supernatant was collected and centrifuged at 2000xg for 20 min to remove debris. The resulting supernatant was then subjected to further centrifugation at 100,000×g for 1 h at 4 °C using a Beckman Coulter Optima L 90K ultracentrifuge. This step was performed to isolate the exosomes from the remaining cellular components. Following the ultracentrifugation, the cells were washed in serum-free medium with 199 HEPES 25mM (Sigma) and underwent an additional round of ultracentrifugation using the same conditions. This process aimed to ensure the purification of the exosomes.

Transmission electron microscopy (TEM)

Exosomes were plated on copper grids, stained with phosphotungstic acid, and then analyzed by TEM. Secondary electronic imaging was used to capture images at a working distance of 15–25 mm and Digital acquisition and analysis were performed using a fast voltage between 20 and 30 kV using a Jeol T300 system.

Flow cytometry

The isothiocyanate-conjugated anti-human monoclonal antibodies used in this study included anti-CD63 (ab18235, Abcam, US) and anti-CD81 (559518, BD Pharmingen). After the extraction of exosomes, they were washed and resuspended in PBS with 3% FBS. The samples were then subjected to testing using a spray assay, specifically the Becton–Dickinson kit from Canada.

Quantification of MSCs derived exosomes

Exosome concentration was measured using the BCA Protein Assay Kit (Novagen) according to the manufacturer’s instructions. The ratio of reagents used was 2:100, 4%Cupric Saltate: BCA solution. Exosome-enriched samples were diluted 1:10 (tenfold) in PBS, supplemented with BCA reagent, and incubated at 37 °C for 30 min before using a NanoDrop™ spectrophotometer (ND-1000, Thermo Fisher Scientific, Wilmington, 1999; 2 measurements. DE, USA) to determine the corresponding absorbance at 562 nm. A standard curve was constructed using the same procedure at bovine serum albumin concentrations ranging from 50 to 250 g/mL.

Purified Exosomes were kept overnight in the medium used for their collection then the pellet was stored at − 80℃.

Loading of vincristine into exosomes using sonication

Vincristine (VCR sulfate) was obtained from a Drug called Vinracine (Hikma, London, United Kingdom) with a concentration of 1mg/ml. to prepare the sample 1ml of VRC was added to 1ml of Exosomes to form a total vol. 2ml of EXO- VCR loading.

Exosome-VCR loading was performed using probe sonication method22. EXO- VCR mixture was sonicated using probe sonication (750 v, power of 20%, 6 cycles were applied by 4-s pulse /2-s pause (average time of 24 s), left to cool on ice for 2 min, and then sonicated again. After that Incubation at 37 °C for 1h without shaking).

Measuring loading capacity

Loading capacity was determined by using a Dialysis tubing cellulose membrane (sigma-Aldrich, Massachusetts, USA) against PBS by adding 2 ml of the mixture after probe-sonication into a cellulose dialysis sac bag that was cut off by scissors then the dialysis bag was sealed properly both from top and bottom and inserted into PBS 7.4 with 50 rpm using a shaking incubator at the room temperature. After 0.5h, the media was collected and replaced with new PBS for another 0.5h. Then the media collected and standard dilutions were filtered by syringe and tested using a Waters 2690 Alliance HPLC system equipped with a Waters 996 photodiode array detector. Standard preparation: Weight 1 mg of each standard to obtain stock solution then serial dilution to obtain conc. of (100,80,60,40,20,10 µg/ml) then filtrated using a 0.22 syringe filter and injected 10 µl. Sample preparation: 1) The sample is then filtrated using a 0.22 syringe filter and injected 10 µl. HPLC analysis conditions were: Column Kromasil C8: 4.6 × 250 mm, 5 µm, Mobile phase: 0.1% Formic acid in water: Acetonitrile, Mode of elution: isocratic, Flow rate: 1ml/min, Temperature: Ambient, Wavelength: 254 nm22.

Cytotoxicity assay

T-47D: Breast ductal carcinoma cell line was obtained from the cell culture unit at Nawah Scientific Inc., (Mokatam, Cairo, Egypt). Cells were cultured in DMEM medium containing 100 mg/mL of streptomycin, 100 units/mL of penicillin, and 10% FBS in a humidified, 5% CO2 atmosphere at 37 °C.

The SRB assay was used to assess the cell viability. Aliquots of cell suspension (5 × 10–3 cells) were incubated in complete water for 24 h in 96-well plates. Another aliquot of different mediums was used to treat the cells. The cells were fixed with 150 L of 10% TCA exchanged for 1 h at 4 °C after 72 h of drug treatment. After the removal of the TCA solution, the cells were washed five times using distilled water. Aliquots of 70 L (0.4% w/v) of SRB solution were added and then incubated for 10 min in a dark atmosphere at room temperature. The plate was cleaned three times with 1% acetic acid and allowed to air dry overnight. The protein-bound SRB stain was then eluted with TRIS 150 L (10 mM), and the absorbance was determined at 540 nm using a BMG LABTECH®- FLUOstar Omega microplate reader (Ortenberg, Germany)23,24.

Flowcytometric analysis of CSCs

The levels of the CD24/CD44 cells in untreated/ treated T47D breast cancer cells were evaluated using flow cytometry with a (FITC)-conjugated CD44antibody (cat no. ab27285; Abcam Inc., UK), and (APC/Cy7)-conjugated CD24 antibody (cat no. ab197137; Abcam Inc., UK). Cells were plated at a density of 6 × 104 in 6-well plates for 24 h before treatment with test compounds for 48 h. Afterward, cells were mechanically harvested, centrifuged, resuspended in 500 µl PBS, and incubated with the conjugated anti-CD44-FITC and anti-CD24- APC/CY7 antibodies in a dark room at ambient temperature for 30 min. After staining, cells were injected via ACEA Novocyte™ flow cytometer (ACEA Biosciences Inc., USA). For each sample, 12,000 events were obtained, and positive FITC and/or APC/CY7 cells were measured by quadrant analysis and calculated using ACEA NovoExpress™ software (ACEA Biosciences Inc., San Diego, CA, USA).

Statistical analysis

The findings from this study were displayed as mean ± SD and evaluated using Microsoft excel version 10 supported with Analysis Toolpak add-in at a significance level of < 0.05. One-way analysis of variance (ANOVA) was used Duncan’s test was carried out to determine the similarities and differences between the control and four treated groups.

Ethical approval

This study was approved by faculty of biotechnology ethics committee, MSA university on 18th of march, 2023. This study was reported according to ARRIVE guidelines and also Animal handling and experimentations were conducted in accordance with the Guidelines of the National Institutes of Health (NIH) regarding the care and use of animals for experimental procedures.