Extracellular vesicles isolated from adipose tissue-derived mesenchymal stromal cells as carriers for Paclitaxel delivery

Abstract

Background: Mesenchymal Stromal Cells (MSC)-derived Extracellular Vesicles (EV) represent innovative tools for drug delivery systems. However, their clinical use is limited by the lack of standardized good manufacturing practice (GMP)-compliant isolation and conservation protocols. In this study, we developed a GMP-compliant protocol for the preparation of MSC-EVs and investigated the feasibility of producing EVs loaded with paclitaxel (PTX) for clinical application as drug products.

Methods: Adipose tissues from 13 donors were used to obtain MSC-EVs via culture supernatant ultracentrifugation. EVs loaded with PTX were manufactured by adding the drug to the culture medium of MSCs before supernatant collection. EV identity was verified in terms of concentration/size, protein content, morphology, and expression of EV surface markers. The anti-proliferative activity, accumulation ability in tumor cells and PTX content, as well as their stability over time, were also evaluated.

Results: High numbers of EV/EV-PTX compliant in terms of integrity/identity were obtained and can be successfully stored for up to one year at -80 °C. Cellular studies have shown that EVs are capable of accumulating in tumor cells and, when loaded with PTX, inhibiting the proliferation of a pleural mesothelioma cell line.

Conclusions: These results support the potential future clinical use of EVs as carriers for drug delivery to improve cancer treatment strategies.

Keywords: Antitumor drug; Drug delivery systems; Extracellular vesicles; Mesenchymal stromal cells; Paclitaxel.

Fig. 1

MSCs morphology. (A) Spindle-shaped morphology of MSCs at P3; (B) MSCs morphology after loading with PTX. Magnification 10X

Fig. 2

Flow cytometry analysis of MSCs. Cells displayed high percentages of the typical MSCs-positive markers and are negative for hematopoietic and endothelial markers

Fig. 3

EV characterization by NTA. Analysis of a representative sample of EV (A) and the corresponding EV-PTX (B). Both EV and EV-PTX showed a single peak and a very homogeneous population

Fig. 4

EV characterization by TEM. EV (A) and EV-PTX (B) morphology by TEM analysis. White dotted squares highlight three representative examples of EV and EV-PTX among many others. Panel C show a high magnification (200 nm) image of an EV, underlining the lipid bilayer structure

Fig. 5

EV characterization by flow cytometry analysis. (A) Both EV and EV-PTX show high expression of the tetraspanins CD9, CD63 and CD81 as well as of the typical MSC markers CD105, CD49e, CD146, CD44, and CD29; (B) comparison of the proportion of EV positive for MSC markers with those positive for platelet markers in terms of normalized CD9/CD63/CD81 median fluorescence intensity (%). MFI, Median Fluorescence Intensity

Fig. 6

Quantitative analysis of accumulation of EVs in MSTO-211 H cells. Cells were seeded and treated 48 h later with EVs at the concentration of 7.2 × 10^7 (20 µL) or 5.4 × 10^6 (1.5 µL) for 4–24 h (Panel A and B, respectively). EV accumulation was evaluated by flow cytometry measuring and quantifying the MFI in panel C. Statistical analysis was performer using ANOVA followed by Bonferroni’s test. P < 0.001 ***

Fig. 7

Stability assessment of the fresh product by flow cytometry analysis. EV samples maintain a similar expression of the typical MSCs surface markers CD105, CD49e, CD44, and CD29 up to 2,5 h after isolation, preserving EV both at room temperature (A) and at + 4 °C (B); a decrease in the expression of the typical MSCs markers was observed 12 h after EV isolation keeping EV at + 4 °C (B), althought the differences were not significant. Similar results were obtained analyzing EV-PTX at room temperature (C) and at + 4 °C (D). MFI, Median Fluorescence Intensity

Fig. 8

Stability assessment of the cryopreserved product by flow cytometry analysis. (A) Analysis of EV samples at time points t ≤ 1, t = 6, t = 12 months after cryoconservation; (B) Analysis of EV-PTX at time points t ≤ 1, t = 6, t = 12 months after cryoconservation. No significant differences were found at different time points both in EV and EV-PTX. MFI, Median Fluorescence Intensity

Fig. 9

Impact of pre-processing storage of EV-containing supernatant-NTA analysis. Analysis of a representative EV sample isolated immediately after supernatants collection (fresh SUP, A) compared with those obtained after the storage of supernatant, supplemented with 1% DMSO (SUP-Cryo-DMSO, B) or alone (SUP-Cryo, C)

Fig. 10

Impact of pre-processing storage of EV-containing supernatant-Flow Cytometry Analysis. Supernatants processed immediately after collection (fresh SUP, blue) show higher expression of the typical MSCs surface markers then both supernatants cryopreserved supplemented of 1% DMSO (SUP Cryo-1% DMSO, green) and alone (SUP Cryo, red) at the temperature of -80 °C for 1 month. Samples cryopreserved alone displayed a significant reduction, in comparison with fresh SUP, in the expression, in terms of MFI, of the markers CD44 (** p < 0,01) and CD29 (* p < 0,05). Statistical analysis was performed using 2way ANOVA followed by Bonferroni’s test. Greenhouse-Geisser method was used to apply corrections for multiple comparisons. MFI, Median Fluorescence Intensity

Fig. 11

Characterization of EV from platelet lysate-NTA analysis. The peak of a representative EV sample from platelet lysate show a very homogeneous population in terms of size

Fig. 12

Characterization of EV from platelet lysate-Flow cytometry analysis Samples show high expression of the typical platelet markers HLA-ABC, CD62P, CD41b, CD42a, CD29 (Panel A), and low expression of the typical MSC markers CD105, CD49e and CD44 (Panel B). MFI, Median Fluorescence Intensity