Red Blood Cell Membrane Vesicles for siRNA Delivery: A Biocompatible Carrier With Passive Tumor Targeting and Prolonged Plasma Residency

Abstract

Background: Despite many advances in gene therapy, the delivery of small interfering RNAs is still challenging. Erythrocytes are the most abundant cells in the human body, and their membrane possesses unique features. From them, erythrocytes membrane vesicles can be generated, employable as nano drug delivery system with prolonged blood residence and high biocompatibility.

Methods: Human erythrocyte ghosts were extruded in the presence of siRNA, and the objects were termed EMVs (erythrocyte membrane vesicles). An ultracentrifugation-based method was applied to select only the densest EMVs, ie, those containing siRNA. We evaluated their activity in vitro in B16F10 cells expressing fluorescent tdTomato and in vivo in B16F10 tumor-bearing mice after a single injection.

Results: The EMVs had a negative zeta potential, a particle size of 170 nm and excellent colloidal stability after one month of storage. With 0.3 nM siRNA, more than 75% gene knockdown was achieved in vitro, and 80% was achieved in vivo, at 2 days PI at 2.5 mg/kg. EMVs mostly accumulate around blood vessels in the lungs, brain and tumor. tdTomato fluorescence steadily decreased in tumor areas with higher EMVs concentration, which indicates efficient gene knockdown. Approximately 2% of the initial dose of EMVs was still present in the plasma after 2 days.

Conclusion: The entire production process of the purified siRNA-EMVs took approximately 4 hours. The erythrocyte marker CD47 offered protection against macrophage recognition in the spleen and in the blood. The excellent biocompatibility and pharmacokinetic properties of these materials make them promising platforms for future improvements, ie, active targeting and codelivery with conventional chemotherapeutics.

Keywords: EPR effect; RNA interference; biomimetic; melanoma; nanomedicine.

Figure 1

(a) Overview of the protocol used for the preparation of DCC-siRNA-EMVs. (b) Size (nm) and RNA concentration (µg/mL) after ultracentrifugation. The data refer to n=3 experiments. The initial concentration of siRNA, before DCC, was 10 µg/mL, resulting in a 67% encapsulation efficiency. (c) Concentrations of protein, lipids, and siRNA among the 5 top fractions. p≤0.01 **.

Figure 2

(a) CryoTEM micrographs of EMVs. The scale bar is 200 nm, and it refers to the insert too. Yellow arrows are pointing at EMVs sitting on the side, appearing as rod-like objects. (b) Relative frequencies of the diameters in the analyzed cryoTEM micrographs. Measurements were carried out on 10 micrographies for each category, for a total of n > 200 measurements per typology. One-way ANOVA was performed to assess the statistical significance of the distributions. p≤0.01 **, p ≤ 0.001 *** (c) Freeze fracture TEM micrograph of empty and DCC-siRNA EMVs. For higher magnification images (left), the scale bar is 50 nm. In the right image, a wider field of DCC-siRNA-EMVs is shown (scale bar = 500 nm). The internal content is recognizable as aggregate material (green arrows).

Figure 3

TEM micrographs of freeze-dried, noncentrifuged samples negatively stained with UA Zero. EMVs appear as discoidal, flat objects. Yellow arrows are pointing at EMVs sitting on the side.

Figure 4

Fluorescence superresolution microscope images of siRNA-EMVs. (a) Wide-field image of DCC-siRNA-EMVs; scale bar = 1 µm. (b) Close-up image of an EMV, Cy-5 and CM-DiI signals colocalized, scale bar = 250 nm; (c) non-DCC-siRNA-EMVs, scale bar = 1 µm.

Figure 5

(a) Percentage of siRNA payload loss over the course of four weeks for DCC and siRNA-EMVs. Data are reported over n=3 replicates. (b) Ultrastructural evolution of negatively stained siRNA-EMVs (left) and DCC-siRNA-EMVs (right). REd arrows indicate pancake-shaped EMVs, which appear as rods via cryo-EM. (c) Size evolution of siRNA-EMVs stored at 4 °C. Above the bars, the percentage of recorded events belonging to a specific population is shown. (d) Percentage of phospholipid weight loss in siRNA-EMVs, stored at 4 °C. (e) Agarose gel (2 % w/v) for free siRNA, DCC-siRNA-EMVs, and non-ultracentrifuged siRNA-EMVs exposed to different concentrations of RNAse A. (f) siRNA retention in siRNA-EMVs after freeze drying. The marked results are significant compared with those of the PBS control for the siRNA concentration. p ≤0.05 * p ≤ 0.01 ** p ≤ 0.001 *** p ≤ 0.0001 ****.

Figure 6

Internalization of DCC-siRNA-EMVs by B16F10, CT26 and NHLF cells over three days. The cell nuclei were labeled with Hoechst 33342 (blue), and the membranes were labeled with MemBrite® Fix 488/515 stain (green). The membranes of EMVs were stained with CM-DiI (Orange), while they were loaded with Cy-5-labeled siRNA (red). The scale bar is 50 µm.

Figure 7

(a) Viability of various cell lines treated with 4.5 µg/mL DCC-siRNA-EMVs; (b) log10 of in vitro tdTomato (fold change) at a final concentration of 0.3 nanomolar. The data are expressed as the means ± SDs. The percentage of knockdowns ± SDs is reported below the graph. p ≤ 0.001 ***, p ≤ 0.0001 ****.

Figure 8

(a) Plasma concentration of the anti-tdTomato siRNA lead strand. For calculation of the injected dose (ID), the mice were assumed to have 1.6 mL of blood. (b) Nanograms of anti-tdTomato siRNA lead strand for each gram of organ, as obtained via interpolation of stem‒loop qPCR data from tissues with a calibration curve (Figure S8).

Figure 9

Distribution of Cy-5/DiO DCC-siRNA-EMVs at different time points in frozen sections of mouse lungs via siRNA-Cy-5 (red) and DiO (green) signals in frozen sections of lungs. Nuclei were labeled with Hoechst 33342 (blue). The scale bar is 100 µm.

Figure 10

Distribution of Cy-5/DiO DCC-siRNA-EMVs at different time points in frozen sections of mouse kidneys via siRNA-Cy-5 (red) and DiO (green) signals. Nuclei were labeled with Hoechst 33342 (blue). The scale bar is 100 µm.

Figure 11

Distribution of Cy-5/DiO DCC-siRNA-EMVs at different time points in frozen sections of the mouse spleen via siRNA-Cy-5 (red) and DiO (green) signals. Nuclei were labeled with Hoechst 33342 (blue). The scale bar is 100 µm.

Figure 12

Summary of the knockdown activity of systemically delivered DCC-siRNA-EMVs. (a) Log10 of the tdTomato fold change in vivo at the 2.5 mg/kg dose compared with the basal expression of the gene in control tumors, as analyzed with the Kruskal‒Wallis nonparametric t-test. p ≤ 0.0001 **** (b) Log10 of the tdTomato median fluorescence intensity variation at 0‒48 hours and 24‒48 hours in control, DCC-siRNA-EMV-, and naked siRNA-treated mice. p≤0.05 *, p≤0.01 ** (c) Tumor tissue sections with blood vessel details at 0 and 48 hours. Red blood cells were unanimously positive in the Cy-5 channel at 48 hours and fainter in the DiO channel. The scale bar is 50 μm.

Figure 13

Distribution of Cy-5/DiO DCC-siRNA-EMVs within B16F10-tdTomato tumors via frozen sections of B16F10-tdTomato tumors at different time points after the injection of DCC-siRNA-EMVs with nuclei labeled with Hoechst 33342 (blue), Vybrant DiO (green), Cy-5 siRNA (red) and tdTomato expression (magenta). The scale bar is 100 µm.

Figure 14

Distribution of Cy-5/DiO DCC-siRNA-EMVs at different time points in frozen sections of the mouse brain via siRNA-Cy-5 (red) and DiO (green) signals. Nuclei were labeled with Hoechst 33342 (blue). The scale bar is 100 µm.