Non-Small-Cell Lung Cancer Regression by siRNA Delivered Through Exosomes That Display EGFR RNA Aptamer

Abstract

Lung cancer is the second most common cancer in both men and women and is the leading cause of cancer death in the United States. The development of drug resistance to commonly used chemotherapeutics in non-small-cell lung cancer (NSCLC) poses significant health risks and there is a dire need to improve patient outcomes. In this study, we report the use of RNA nanotechnology to display ligand on exosome that was loaded with small interfering RNA (siRNA) for NSCLC regression in animal trials. Cholesterol was used to anchor the ligand targeting epidermal growth factor receptor on exosomes that were loaded with siRNA to silence the antiapoptotic factor survivin. The cytosolic delivery of siRNA overcame the problem of endosome trapping, leading to potent gene knockdown, chemotherapy sensitization, and tumor regression, thus achieving a favorable IC₅₀ of 20 nmol/kg siRNA encapsulated by exosome particles in the in vivo gene knockdown assessment.

Keywords: EGFR; RNA nanotechnology; aptamer; exosomes; non-small-cell lung cancer; siRNA.

FIG. 1.

Assembly of exosome vesicle loaded with siRNA and RNA nanotechnology decoration. (A) Formulation scheme of EGFR_Apt displaying exosomes loaded with siRNA. (B) Size distribution of exosomes. Purified exosomes were analyzed by NTA with peak size at 109 nm and average size of 177 ± 6 nm. (C) Surface marker characterization. Purified exosomes from four independent purification were assayed by immunoblotting to confirm exosome surface marker CD63 and TSG 101. (D) Design of EGFR_Apt arrowtail pRNA nanoparticles for ligand displaying. (E) Assembly of EGFR_Apt arrowtail pRNA nanoparticles assay by 10% Native PAGE in TBE buffer stained by EtBr. (F) Loading efficiency of siRNA into exosomes assayed by comparing fluorescent intensity in unloaded supernatant of siRNA loading with Exo and without Exo. EGFR, epidermal growth factor receptor; EtBr, ethidium bromide; NTA, nanoparticle tracking analysis; PAGE, polyacrylamide gel electrophoresis; pRNA, packaging RNA; siRNA, small interference RNA; TBE, tris/borate/EDTA buffer.

FIG. 2.

Design and assembly of si-survivin harboring 3WJ RNA nanoparticle. Design of RNA nanoparticle shown on the left panel, 2′ F-modified sequence in lower case. Self-assembly of RNA nanoparticles was verified by 12% TBE PAGE, EtBr staining channel in green, Alexa₆₄₇ channel in red (right panel). 3WJ, 3-way-junction.

FIG. 3.

Protein expression level in different NSCLC cell lines and a human bronchial epithelial cell line. Western blot was conducted to screen the protein expression level of EGFR receptor (upper panel) and survivin (lower panel). NSCLC, non-small-cell lung cancer.

FIG. 4.

EGFR_Apt/Exo/siSurvivin suppresses survivin expression in human lung cancer. (A) Cellular uptake of siSurvivin delivered by EGFR_Apt/Exo and Exo only compared by flow cytometry on both H596 and H1568 NSCLC cell lines, **P < 0.01, ***P < 0.001 by Student's t-test, n = 3. Raw data and gate setting in Supplementary Fig. S4. (B) mRNA level of survivin gene quantified by qRT-PCR assay post 48 h treatment of EGFR_Apt/Exo/siSurvivin, EGFR_Apt/Exo/siCtr, Exo/siSurvivin, and TES buffer in 50 nM of siRNA concentration on both H596 and H1568 NSCLC cell lines. **P < 0.01, ****P < 0.0001 by Student's t-test, n = 3. (C) Protein expression level of Survivin evaluated by western blot post 48 h treatment of EGFR_Apt/Exo/siSurvivin, EGFR_Apt/Exo/siCtr, Exo/siSurvivin and TES buffer in 50 nM of siRNA concentration on both H596 and H1568 NSCLC cell line. qRT-PCR, real-time quantitative reverse transcription polymerase chain reaction; TES, tris/EDTA/saline buffer.

FIG. 5.

Silencing of survivin suppressed tumor cell growth and sensitized cisplatin cytotoxicity in NSCLC Cells. (A) IncuCyte cell proliferation assay on both H596 and H1568 NSCLC cell lines posttreating with EGFR_Apt/Exo/siSurvivin, EGFR_Apt/Exo/siCtr, Exo/siSurvivin, and TES buffer in 50 nM of siRNA concentration. n = 3, error bar ± SEM. (B) Cell apoptosis evaluation by Annexin V-positive cells using flow cytometry on both H596 and H1568 NSCLC cell lines posttreating with EGFR_Apt/Exo/siSurvivin, EGFR_Apt/Exo/siCtr, Exo/siSurvivin, and TES buffer in 50 nM of siRNA concentration for 48 h followed by 24 h treatment with 5 μM cisplatin, n = 3, *P < 0.05, **P < 0.01, error bar ± SD. Original data and gating refer to Supplementary Fig. S4. (C) Cisplatin-induced apoptosis marker evaluation by western blot on both H596 and H1568 NSCLC cell lines posttreating with EGFR_Apt/Exo/siSurvivin, EGFR_Apt/Exo/siCtr,/Exo/siSurvivin, and TES buffer in 50 nM of siRNA concentration for 48 h followed by 24 h treatment with or without 5 μM cisplatin. SD, standard deviation; SEM, standard error of the mean.

FIG. 6.

In vivo evaluation of EGFR_Apt/Exo/siSurvivin on NSCLC tumor xerograph mice. (A) Biodistribution study by tracing Alexa₇₅₀-labeled siSurvivin delivered by exosomes. H596 NSCLC cell-developed tumor xenograft mice were I.V. administrated with 100 μL of 20 μM of RNA Alexa₇₅₀-labeled siSurvivin loaded in exosomes or EGFR_Apt/Exo and imaging by IVIS post 1, 4, 8, 24, 32, and 48 h shown on the left. Organ was dissected at 48 h and imaged by IVIS shown on the right. (B) Scheme of exosome injection and tumor isolation time frame for dose optimization study. (C) mRNA level of survivin gene in tumor quantified by qRT-PCR assay post three repeated I.V. administration of EGFR_Apt/Exo/siSurvivin in 4, 10, 20, and 40 nmol/kg of siRNA concentration and TES buffer on H596 NSCLC cell-developed tumor xenograft mice, n = 3, error bar ± SD. Protein level assayed by western blot shown in (D). (E) Tumor growth curve traced during six repeated I.V. administration of EGFR_Apt/Exo/siSurvivin, EGFR_Apt/Exo/siCtr, Exo/siSurvivin, and TES buffer in 40 nmol/kg of siRNA concentration on H596 NSCLC cell-developed tumor xenograft mice. ***P < 0.001, ****P < 0.0001, two-way ANOVA (mix model repeated measurement), n = 5, error bar ± SEM. ANOVA, analysis of variance; I.V., intravenus; IVIS, in vivo imaging system; ns, no significance.