Article Figures & Data
Figures
- Figure 1.
Schematic of IVT EXO-DEPT preparation and use, involving several steps. (1) Generation of IVT HChrR6 mRNA. (2) Its transfection into HEK293 cells in the presence of polyethylenimine. (3) Confirmation that EVs generated by loaded HEK293 producer cells contain HChrR6 mRNA. (4) Conversion of loaded EVs into IVT EXO-DEPTs by incubation with purified EVHB protein. (5) IVT EXO-DEPT–mediated delivery of HChrR6 mRNA to BT474 cells. (6) Addition of the prodrug CNOB or CB1954 (tretazicar). (7) Prodrug conversion within the cells into the drug. (8) Cell death.
- Figure 2.
IVT HChrR6 mRNA functionality and its amount in IVT EXO-DEPTs. A, Test of the functionality of IVT HChrR6 mRNA. Its translated product converts CNOB into MCHB as measured from MCHB fluorescence. Increasing amounts of the translated HChrR6 mRNA protein (quantified by Bradford assay) generated increasing fluorescence (n = 3). Numbers in the abscissa denote: 1, PBS; 2, CNOB 15 μmol/L; 3, NADPH 1 mmol/L; 4, full reaction mix (Rx) without HChrR6 protein. (CNOB was 15 μmol/L; NADPH, 1 mmol/L); 5, Rx + 2.5 ng protein; 6, Rx + 5 ng protein; 7, Rx + 10 ng protein; 8, Rx + 20 ng protein; 9, Rx + 40 ng protein; 10, Rx + 80 ng protein; and 11, Rx + 160 ng protein. B, The IVT EXO-DEPTs contain more HChrR6 mRNA compared with P EXO-DEPTs: <50 of the former (red bar), but 5,000 of the latter (blue bar) are needed to donate one mRNA copy (n = 3).
- Figure 3.
A, IVT EXO-DEPTs interaction with HER2+ BT474 cells and HChrR6 mRNA delivery. Flow cytometry results of 1.62 × 106 BT474 cells incubated for 6 hours (top), or 24 hours (bottom) with PKH26-labeled (red) or unlabeled (yellow) 5 × 1010 IVT EXO-DEPTs. At 6 hours, 0.6% cells bounded the EVs (red; top, right); at 24 hours this number increases to 80% (bottom, right; n = 3). B, HChrR6 mRNA copy number in BT474 cells after 6-hour incubation with IVT EXO-DEPTs (red bar). No HChrR6 mRNA was detected in BT474 cells incubated for the same duration with directed EVs not containing the IVT mRNA (*, P < 0.05; n = 3); consequently, no blue bar (representing unloaded EVs) is seen in the figure.
- Figure 4.
Comparative efficacy of IVT- and P EXO-DEPTs. Comparison of the kinetics of HChrR6 gene expression by BT474 cells (104) receiving the HChrR6 mRNA by IVT EXO-DEPTs (red bars) or by P EXO-DEPTs (blue bars), as determined by the MCHB test. The number of the two kinds of the EVs was adjusted to deliver 104 mRNA copies. This required 3 × 105 IVT- and 5 × 107 P EXO-DEPTs (*, P < 0.05; **, P < 0.01; ***, P < 0.001; n = 3). The IVT EXO-DEPTs generate superior results.
- Figure 5.
In vivo effectiveness of the IVT EXO-DEPTs. A, Administration schedule; the EXO-DEPT numbers and the tretazicar amount are for each dose per mouse. B, Volume of orthotopically implanted BT474 HER2+ xenografts in mice, as measured by a caliper; each data point is mean value for individual treatment groups (P < 0.001). The four groups of mice used are identified in the figure. C, Box and Whisker plot of the growth rate of the tumors (***, P < 0.001; n = 7). Mice receiving full treatment (IVT EXO-DEPTs + tretazicar) show near-complete arrest of xenograft growth. i.p. intraperitoneal.
- Figure 6.
Hematology and serum chemistry. A, Hematology of treated (red) and nontreated (blue) mice. B, Serum chemistry of treated and nontreated mice. Abscissa abbreviations for the constituents are standard; they are spelled out in full in Supplementary Table S1, which also provides numerical values. Mice receiving full treatment show essentially no off-target injury.
Additional Files
Supplementary Data
- Supplementary Data - Supplementary Figure S1 shows composition of the EVHB protein and making of EXO-DEPTS. Supplementary Figure S2 shows the size and proteins that characterize extracellular vesicles. Supplementary Figure S3 shows that the mRNA is inside the extracellular vesicles. Supplementary figure S4 shows that low doses of IVT EXO-DEPTs are ineffective. Supplementary Table S1 provides full names of abbreviations used in Figure 6.
Volume 19, Issue 3
