Development of Artificial Plasma Membranes Derived Nanovesicles Suitable for Drugs Encapsulation

Abstract

Extracellular vesicles (EVs) are considered as promising nanoparticle theranostic tools in many pathological contexts. The increasing clinical employment of therapeutic nanoparticles is contributing to the development of a new research area related to the design of artificial EVs. To this aim, different approaches have been described to develop mimetic biologically functional nanovescicles. In this paper, we suggest a simplified procedure to generate plasma membrane-derived nanovesicles with the possibility to efficiently encapsulate different drugs during their spontaneously assembly. After physical and molecular characterization by Tunable Resistive Pulse Sensing (TRPS) technology, transmission electron microscopy, and flow cytometry, as a proof of principle, we have loaded into mimetic EVs the isoquinoline alkaloid Berberine chloride and the chemotherapy compounds Temozolomide or Givinostat. We demonstrated the fully functionality of these nanoparticles in drug encapsulation and cell delivery, showing, in particular, a similar cytotoxic effect of direct cell culture administration of the anticancer drugs. In conclusion, we have documented the possibility to easily generate scalable nanovesicles with specific therapeutic cargo modifications useful in different drug delivery contexts.

Keywords: drugs-delivery; extracellular vesicles; nanomedicine.

Figure 1

Analysis of T98G-derived EVs size and concentration. (A) EVs isolated by ultracentrifugation and (B) by plasma membranes reassembling protocol (M-NVs) were analysed through TRPS (qNano Gold, Izon). (C) Histogram of size distribution values of minimum, maximum, mean and mode values of the analysed nanoparticles (n = 2000).

Figure 2

TEM ultrastructural analysis (A,B), size and concentration (C,D) and z-charge (E,F) comparison between membranes reassembling mimetic nanovesicles (M-NVs) and exosomes both isolated form T98G cells, determined through TRPS analysis (qNano Gold, Izon).

Figure 3

CD63 expression in R1 and R2 sub-populations from T98G exosomes (A) and M-NVs (B) by Amnis ImageStreamX MarkII flow cytometer. Examples of CD63 + and CD63- (Ch.5) EVs (60×) are reported (C). BF = Brightfield; SSC = scatterplot. A total of 25,000 particles were acquired.

Figure 4

Amnis ImageStreamX MarkII flow cytometry and fluorescence microscope analysis of M-NVs stained with the red fluorescent PHK26 dye (A). PHK26 decorated M-NVs and unstained M-NVs were then separately administered to growing T98G cells. Cells were fixed after 24 h, DAPI stained and visualized under Nikon Eclipse TS100 fluorescence microscope (B) (100×, scale bars = 10 µm). For cytometry, a total of 25,000 particles were acquired.

Figure 5

Kinetics of internalization of M-NVs stained with PHK67 dye into T98G cells at different time intervals (i.e., 0.5–1–2–24 h) visualized by inverted fluorescence microscope (40× magnification). Scale bar = 10 µm.

Figure 6

Fluorescence microscope and flow cytometric examinations of T98G cells after administration of electroporated membranes-generated vesicles (M-NVs, ~2 × 10⁷ particles) with pEGFP vector (0.5 µg). Cells (~2 × 10³) were fixed after 72 h p.t, DAPI stained and visualized by fluorescence microscope (100× magnification, scale bars = 10 µm) (A). Amnis ImageStreamX MarkII flow cytometry analysis was used to quantify the uptake efficiency (as percentage of fluorescent cells) of pEGFP electroporated M-NVs (~2 × 10⁷ particles) into T98G cells (~5 × 10⁶). As a result, R1 single cells subpopulation (n = 5000) showed a 94.5% of green fluorescent signals (R2). R2 and R3 representative cells were visualized using 40× magnification (B).

Figure 7

Amnis ImageStreamX MarkII flow cytometry analysis of membranes generated vesicles (M-NVs) stained with green fluorescent PHK67 dye and encapsulated with an Alexa Fluor 633 conjugated secondary antibody, visualized in Brightfield (BF), Ch.2 and Ch.5 fluorescent channels, respectively at 60× magnification. For cytometry, a total of 15,000 particles were acquired.

Figure 8

Amnis ImageStreamX MarkII flow cytometry analysis of T98× cells (R2 gates = 5000 single cells each) after administration of plasma membranes generated nanovesicles (M-NVs) (A) or with Berberine (BBR) encapsulated M-NVs, visualized in Brightfield (BF) and Ch.2 fluorescent channels (40×) (B). The percentage of BBR-positive M-NVs were evaluated by flow cytometry (i.e., 87.4% of R2 gated cells, C left panel); these M-NVs were then administered overnight to growing T98G cells and observed using Nikon Eclipse TS100 inverted fluorescent microscope (C, right panel) (40×, scale bar = 10 µm).

Figure 9

Cytofluorimetric viability assays in three different human astrocytoma cell lines (i.e., T98G, U138-MG and Res259) at 24 h after direct administration of mock plasma membranes generated vesicles (MVs), Temozolomide (TMZ) or Givinostat (GVS) and M-NVs loaded with TMZ or GVS separately (each 50 µM). Experiments were in triplicates analyzing 5000 cells for each treatment and asterisks indicate p < 0.05, Anova One-way compared to mock samples.

Figure 10

Flow cytometry analysis of apoptotic induction. (A) Annexin V expression in T98G cells after 24-h administration of mock membranes-derived vesicles (M-NVs), Temozolomide (TMZ 50 µM) and M-NVs encapsulated with equal TMZ concentration (L = live; D = dead; EA = early apoptotic; LA = late apoptotic cells percentages). Microscope contrast phase images (magnifications, 10× and 40×, scale bars = 10 µm). (B) Summary histogram of apoptosis vs. live percentages according to Annexin V assay. Experiments were in triplicates analyzing 5000 cells for each treatment and asterisks indicate p < 0.05 Anova One-way compared to mock M-NVs samples.