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. 2018 Apr 8:2018:7053623.
doi: 10.1155/2018/7053623. eCollection 2018.

Evaluation of Cytochalasin B-Induced Membrane Vesicles Fusion Specificity with Target Cells

Marina Gomzikova  1 , Sevindzh Kletukhina  1 , Sirina Kurbangaleeva  1 , Albert Rizvanov  1
Affiliations

Evaluation of Cytochalasin B-Induced Membrane Vesicles Fusion Specificity with Target Cells

Marina Gomzikova et al. Biomed Res Int. .

Abstract

Extracellular vesicles (EV) represent a promising vector system for biomolecules and drug delivery due to their natural origin and participation in intercellular communication. As the quantity of EVs is limited, it was proposed to induce the release of membrane vesicles from the surface of human cells by treatment with cytochalasin B. Cytochalasin B-induced membrane vesicles (CIMVs) were successfully tested as a vector for delivery of dye, nanoparticles, and a chemotherapeutic. However, it remained unclear whether CIMVs possess fusion specificity with target cells and thus might be used for more targeted delivery of therapeutics. To answer this question, CIMVs were obtained from human prostate cancer PC3 cells. The diameter of obtained CIMVs was 962,13 ± 140,6 nm. We found that there is no statistically significant preference in PC3 CIMVs fusion with target cells of the same type. According to our observations, the greatest impact on CIMVs entry into target cells is by the heterophilic interaction of CIMV membrane receptors with the surface proteins of target cells.

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Figures

Figure 1
Figure 1
The morphology and content of CIMVs. Brightfield (a), Hoechst 33342 (b), and Calcein AM staining (c) were used to analyze the morphology and presence of nuclear and cytoplasmic components, respectively, in CIMVs.
Figure 2
Figure 2
Size distribution of PC3 CIMVs. (a) Flow cytometry data. Light gray peak: distribution of PC3 CIMVs by forward-scatter (FSC); dark gray peaks: size calibration beads of 0.22, 0.45, 0.88, 1.34, and 3.4 μm. (b) Dynamic light scattering data. Curves of 3 parallel measurements are represented.
Figure 3
Figure 3
Interaction of CIMVs with recipient cells. Fluorescent microscopy (a)–(c) of PC3 recipient cells 4 h after 10 μg/ml CIMVs application was carried out. Recipient cells were prelabeled with DiO cytoplasmic membrane dye and CIMVs were prelabeled with DiD membrane dye. Scale bar: 5 μm.
Figure 4
Figure 4
The fusion efficiency of PC3 CIMVs with PC3, SH-SY-5Y, HCT116, and HeLa cell lines (flow cytometry data). Dark gray column: PC3 CIMVs interaction with PC3 target cells (homophilic interaction). ∗p < 0.05 indicates significance level.
Figure 5
Figure 5
Effect of low-temperature (4°С) incubation and proteinase K treatment on fusion efficiency of CIMVs with target cells. Results are shown as percentage relative to positive control (100%, cells incubated with CIMVs at 37°C in full medium).
Figure 6
Figure 6
The fusion efficiency of HeLa CIMVs with PC3, SH-SY-5Y, HCT116, and HeLa cell lines (flow cytometry data). Dark gray column: HeLa CIMVs interaction with HeLa target cells (homophilic membrane proteins interaction). ∗p < 0.05 indicates significance level.
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