Delivery of RNA nanoparticles into colorectal cancer metastases following systemic administration

Abstract

The majority of deaths from all cancers, including colorectal cancer (CRC), is a result of tumor metastasis to distant organs. To date, an effective and safe system capable of exclusively targeting metastatic cancers that have spread to distant organs or lymph nodes does not exist. Here, we constructed multifunctional RNA nanoparticles, derived from the three-way junction (3WJ) of bacteriophage phi29 motor pRNA, to target metastatic cancer cells in a clinically relevant mouse model of CRC metastasis. The RNA nanoparticles demonstrated metastatic tumor homing without accumulation in normal organ tissues surrounding metastatic tumors. The RNA nanoparticles simultaneously targeted CRC cancer cells in major sites of metastasis, such as liver, lymph nodes, and lung. Our results demonstrate the therapeutic potential of these RNA nanoparticles as a delivery system for the treatment of CRC metastasis.

Keywords: RNA nanoparticle; RNA therapeutics; bacteriophage phi29; colorectal cancer; metastasis; nanobiotechnology.

Figure 1. Construction of multi-module pRNA nanoparticles for CRC metastasis targeting

a. 2D sequence of pRNA 3WJ domain composed of three RNA oligomers (a, b, c), folic acid (FA) and near-infrared fluorescent dye Alexa647 (left); 3D model and AFM images of pRNA 3WJ motif harboring monomeric pRNA as functional modules. b. The most common sites of CRC metastasis—liver, lung and lymph nodes. The FA-conjugated pRNA (FA-pRNA) nanoparticle targets FRα positive tumors upon systemic administration. c. CRC liver, lung and lymph node experimental metastasis modes (Green: metastatic tumors)

Figure 2. Analysis of FRα expression in CRC liver and lung metastasis

a Examples of immunohistochemical staining for FRα in liver and lung CRC metastases. Positive FRα staining of CRC was cytoplasmic or membranous or both; most positive cases showed both patterns. FRα staining was negative in normal liver and lung tissues. b. Differences in proportion of positive cells and intensity of staining were noted in positively stained cases and formed the basis of our grading system. Comprehensive total score that weighs both factors was calculated by summation of proportion and intensity values. High FRα expression (score 3-6) was detected in 63% of primary CRCs, 81% of CRC liver metastases and 60% of CRC lung metastasis.

Figure 3. FA-pRNA nanoparticles binding to CRC cells

a. Binding and entry of FA-pRNA-Alexa647 nanoparticles into KM20 and HT29 cells in vitro. Magnification 40×. b. Single dose (4μg/g in 100 μl of PBS) of FA-pRNA-Alexa647 labeled nanoparticles was administered intravenously into mice with HT29 liver metastases. Accumulation of fluorescently-labeled nanoparticles was evaluated microscopically 2h after RNA nanoparticle administration. Yellow arrow: pRNA-Alexa647 (top panel), FA-pRNA-Alexa647 (bottom panel); green: GFP-expressing cancer cells; blue: DAPI stain for nuclear dsDNA; magenta: Alexa647. Magnification 40×.

Figure 4. FA-pRNA nanoparticles delivery into CRC liver metastases

a. pRNA-Alexa647 and FA-pRNA-Alexa647 labeled nanoparticles were administered intravenously (single dose of 1 μg/g in 300μl of PBS) every 2h (three doses total) into mice with KM20 (left panel) and HT29 (right panel) liver metastases and evaluated macroscopically 6h after the first administration. b. Accumulation of fluorescently-labeled nanoparticles was evaluated microscopically in KM20 (left panel) and HT29 (right panel) and in normal liver adjacent to KM20 and HT29 metastases, 6h after the first pRNA dose administration. Control: PBS; pRNA-Alexa647 treated mice. Green: GFP-expressing cancer cells; blue DAPI; magenta: Alexa647. Magnification 40×.

Figure 5. FA-pRNA nanoparticle delivery into CRC lung and lymph node metastases

a. Mice with KM20 lung and lymph node metastases were treated with pRNA-Alexa647 and FA-pRNA-Alexa647 (single dose of 1 μg/g in 500 μl of PBS) every 2h (2 doses total). Accumulation of fluorescently-labeled nanoparticles was evaluated macroscopically in mice with KM20 lung and lymph node metastases 4h after the first pRNA dose administration. b. Confocal imaging of KM20 lung and lymph node metastases. Control: pRNA-Alexa647 treated mice. Green: GFP-expressing cancer cells; blue: DAPI; magenta: Alexa647. Magnification 40×