Nanoparticle orientation to control RNA loading and ligand display on extracellular vesicles for cancer regression

Abstract

Nanotechnology offers many benefits, and here we report an advantage of applying RNA nanotechnology for directional control. The orientation of arrow-shaped RNA was altered to control ligand display on extracellular vesicle membranes for specific cell targeting, or to regulate intracellular trafficking of small interfering RNA (siRNA) or microRNA (miRNA). Placing membrane-anchoring cholesterol at the tail of the arrow results in display of RNA aptamer or folate on the outer surface of the extracellular vesicle. In contrast, placing the cholesterol at the arrowhead results in partial loading of RNA nanoparticles into the extracellular vesicles. Taking advantage of the RNA ligand for specific targeting and extracellular vesicles for efficient membrane fusion, the resulting ligand-displaying extracellular vesicles were capable of specific delivery of siRNA to cells, and efficiently blocked tumour growth in three cancer models. Extracellular vesicles displaying an aptamer that binds to prostate-specific membrane antigen, and loaded with survivin siRNA, inhibited prostate cancer xenograft. The same extracellular vesicle instead displaying epidermal growth-factor receptor aptamer inhibited orthotopic breast cancer models. Likewise, survivin siRNA-loaded and folate-displaying extracellular vesicles inhibited patient-derived colorectal cancer xenograft.

Figure 1. RNA nanotechnology for decorating native EVs

(a) AFM image of extended 3WJ of the motor pRNA of bacteriophage phi29. (b) Illustration of the location for cholesterol labeling of the arrow-head or arrow-tail of 3WJ. (c) Negative-stained EM image of EVs from HEK293T cells purified with differential ultracentrifugation method and cushion modified ultracentrifugation method. (d–g) NTA for size analysis and DLS for Zeta potential measurements. (h) 2D structure (left panel) and native PAGE for testing 3WJ assembly from three component strands, as indicated. (i). EVs loading and RNA aptamer display.

Figure 2. Comparison of the role between arrow-head and arrow-tail 3WJ

(a–b) Illustration showing the difference between arrow-head and arrow-tail display. (c) Syner gel to test arrow-head and arrow-tail Alexa₆₄₇-3WJ/EV degradation by RNase in FBS. The gel was imaged at Alexa₆₄₇ channel (d) and the bands were quantified by Image J. (e–i) Assay to compare cell binding of folate-3WJ arrow-tail (e–g) and arrow-head (h–i) on folate receptor positive and negative cells.

Figure 3. Specific binding and siRNA delivery to cells in vitro using PSMA aptamer-displaying EVs

(a) Flow cytometry (left) and confocal images (right) showing the binding of PSMA RNA aptamer-displaying EVs to PSMA-receptor positive and negative cells. Nucleus (Blue), cytoskeleton (Green), and RNA (Red) in confocal images. (b) RT-PCR assay for PSMA aptamer-mediated delivery of survivin siRNA by EVs to PSMA(+) prostate cancer cells. Statistics: n=4; experiment was run in four biological replicates and two to four technical repeats with an ANOVA analysis; holm adjusted p = 0.0120, 0.0067 comparing PSMA_apt/EV/siSurvivin to PSMA_apt/EV/siScramble and 3WJ/EV/siSurvivin, respectively. (c) MTT assay showing reduced cellular proliferation. n=3, p = 0.003, 0.031 comparing PSMA_apt/EV/siSurvivin to PSMA_apt/EV/siScramble and 3WJ/EV/siSurvivin respectively. *p<0.05, **p<0.01.

Figure 4. Animal trials using ligands displaying EV for tumor inhibition

(a) Organ images showing specific tumor targeting 8 hrs after systemic injection of folate displaying EVs to mice with subcutaneous KB cell xenografts. n = 2, two independent experiments. (b) Intravenous treatment of nude mice bearing LNCaP-LN3 subcutaneous xenografts with PSMA_apt/EV/siSurvivin or PSMA_apt/EV/siScramble (both with 0.6 mg/kg, siRNA/mice body weight), and PBS, injected twice per week for three weeks. n=10 biological replicates, 2 independent experiments, and statistics were calculated using a two-sided t-test expressed as averages and with standard deviation. p = 0.347, 0.6–2, 1.5e–6, 8.2e–8, 2.1e–7, 1.0e–7, 1.9e–7, 1.8e–6 for days 15, 18, 22, 25, 29, 32, 36, and 39 respectively for PSMA_apt/EV/siSurvivin compared to control. (c) Body weight of mice during the time course of EVs treatment. (d) RT-PCR showing the trend of knockdown survivin mRNA expression in prostate tumors after EV treatment.

Figure 5. EGFR aptamer displaying EVs can deliver survivin siRNA to breast cancer orthotopic xenograft mouse model

(a) EGFR aptamer displaying EVs showed enhanced targeting effect to breast tumor in orthotopic xenograft mice models. (b) Intravenous treatment of nude mice bearing breast cancer orthotopic xenografts with EGFR_apt/EV/siSurvivin and controls (n=5). After 6 weeks, EGFR_apt/EV/siSurvivin treated group had significantly smaller tumor size than other controls. p = 0.008 comparing EGFR_apt/EV/siSurvivin to EGFR_apt/EV/siScramble. (c) Analysis of the protein expression in tumor extracts showed that EGFR_apt/EV/siSurvivin treatment significantly reduced the expression of Survivin. p=0.0004 comparing EGFR_apt/EV/siSurvivin to EGFR_apt/EV/siScramble. (d) Quantitative real-time PCR on extracted RNA from tumors showed the reduction of Survivin mRNA in the EGFR_apt/EV/siSurvivin treated mice compared to controls. p=0.024 comparing EGFR_apt/EV/siSurvivin to EGFR_apt/EV/siScramble. Error bars indicate s.e.m. * p < 0.05, ** p < 0.01, *** p<0.001.

Figure 6. Folate displaying EVs can deliver survivin siRNA to patient derived colorectal cancer xenograft (PDX-CRC) mouse model

(a) Intravenous treatment of nude mice bearing PDX-CRC xenografts with FA/EV/siSurvivin and controls (n=4). After 6 weeks, FA/EV/siSurvivin treated group had significantly smaller tumor size, p = 0.0098 and 0.0387 comparing FA/EV/siSurvivin to FA/EV/siScramble at week 4 and week 5 respectively. (b) Lower tumor weight than controls. p = 0.0024 comparing FA/EV/siSurvivin to FA/EV/siScramble. Error bars indicate s.e.m. * p < 0.05, ** p < 0.01.