Cell-Derived Vesicles for in Vitro and in Vivo Targeted Therapeutic Delivery

Abstract

Efficient delivery of therapeutics across the cell membrane to the interior of the cell remains a challenge both in vitro and in vivo. Here, we demonstrate that vesicles derived from cellular membranes can be efficiently loaded with cargo that can then be delivered to the interior of the cell. These vesicles demonstrated cell-targeting specificity as well as the ability to deliver a wide range of different cargos. We utilized this approach to deliver both lipophilic and hydrophilic cargos including therapeutics and DNA in vitro. We further demonstrated in vivo targeting and delivery using fluorescently labeled vesicles to target tumor xenografts in an animal. Cell-derived vesicles can be generated in high yields and are easily loaded with a variety of cargos. The ability of these vesicles to specifically target the same cell type from which they originated provides an efficient means of delivering cargo, such as therapeutics, both in vitro and in vivo.

Figure 1

Cell-derived vesicle characterization. (A) Wide-field fluorescence image of vesicles loaded with the fluorescent dye fluorescein. (B) Fluorescence correlation spectroscopy correlogram of vesicles used to determine vesicle concentration and relative yield. (C) Plot of vesicle size distribution at different cavitation pressures as determined by dynamic light scattering.

Scheme 1. Schematic of Vesicle Generation, Loading, and Isolation

Cultured cells undergo nitrogen cavitation in the presence of cargo in free solution followed by serial centrifugation to generate purified vesicles. Vesicles serve as nanocarriers for hydrophilic cargo encapsulated during cavitation on the interior or for lipophilic cargo that can be embedded in the vesicle membrane.

Figure 2

Cell-targeting specificity. (A) Comparison of HEK vesicles delivered to HEK cells (black) versus HEK vesicles delivered to A549 cells (red). (B) Comparison of RAW vesicles delivered to RAW cells (black) versus RAW vesicles delivered to A549 cells (red). (C) Comparison of HCT vesicles delivered to HCT cells (black) versus RAW vesicles delivered to HCT cells (red). (D) Wide-field fluorescence image of DiI-labeled RAW vesicles delivered to RAW cells after 2.5 h showing clear loading. (E) Wide-field fluorescence image of DiI-labeled RAW vesicles delivered to A549 cells after 2.5 h showing limited cellular uptake. Norm ID is the integrated density of the image normalized to the time 0 value. Each data point is the average of five experiments. A Student’s t-test was used to determine significance between end points. Each end point was significant with a p value of <.001.

Figure 3

Efficacy of cisplatin-loaded vesicles. Comparison of cell growth at time 0, 24, 48, and 72 h for A549 cells with no treatment (black), treated with empty vesicles (gray), with cisplatin-loaded vesicles (purple), and free cisplatin in solution (pink). Empty vesicles have no effect on cell growth while both free cisplatin and loaded vesicles show similar efficacy in killing A549 cells. Each data point is the average of five experiments. A Student’s t-test was used to determine significance. The asterisk “*” indicates a p value of <.001.

Figure 4

Vesicle-based gene delivery. (A) Wide-field image of HEK cells in the absence of plasmid. (B) Wide-field image of HEK cells after exposure to Dendra2 plasmid-loaded vesicles showing clear cellular delivery based on the expression of the fluorescent protein.

Figure 5

Confocal imaging of vesicle delivery. Confocal image of HEK 293 cells after the delivery of vesicles loaded with fluorescein (interior) and DiD (cell membrane). The interior of the cell is filled with fluorescein and vesicles can be seen on the cell surface.

Figure 6

Mice bearing A549 xenografts on the right shoulder (dashed blue ovals) were injected with (A) dye (DiR) alone, (B) dye-labeled vesicles derived from HEK cells, (C) dye-labeled vesicles derived from A549 cells, and (D) dye-labeled vesicles derived from RAW264.7 cells demonstrating RAW vesicles specifically targeted the A549 xenograft. Free domain photograph courtesy of JRM.