RNA Nanotechnology for Codelivering High-Payload Nucleoside Analogs to Cancer with a Synergetic Effect

Abstract

Nucleoside analogs are potent inhibitors for cancer treatment, but the main obstacles to their application in humans are their toxicity, nonspecificity, and lack of targeted delivery tools. Here, we report the use of RNA four-way junctions (4WJs) to deliver two nucleoside analogs, floxuridine (FUDR) and gemcitabine (GEM), with high payloads through routine and simple solid-state RNA synthesis and nanoparticle assembly. The design of RNA nanotechnology for the co-delivery of nucleoside analogs and the chemotherapeutic drug paclitaxel (PTX) resulted in synergistic effects and high efficacy in the treatment of Triple-Negative Breast Cancer (TNBC). The 4WJ-drug complexes were confirmed to have efficient tumor spontaneous targeting and no toxicity because the motility of RNA nanoparticles has been previously shown to enable these RNA-drug complexes to spontaneously accumulate in tumor blood vessels. The negative charge of RNA enables those RNA complexes that are not targeted to tumor vasculature to circulate in the blood and enter the urine through the kidney glomerulus, without accumulating in organs, therefore being nontoxic. Drug incorporation into RNA 4WJ can be precisely controlled with a defined loading amount, location, and ratio. The incorporation of nucleoside analogs into 4WJ only requires one step using nucleoside analogue phosphoramidites during solid-phase RNA synthesis, without the need for additional conjugation and purification processes.

Keywords: RNA nanotechnology; cancer therapy; combinational therapy; drug delivery; floxuridine; gemcitabine; nucleoside analog.

Fig. 1.. Construction of 4WJ RNA nanoparticles carrying different nucleoside analogues.

(A) Chemistry structure of FUDR (left); schematics of incorporating FUDR into RNA strands via solid-phase synthesis (middle); PAGE gel verifying the assembly of 4WJ-FUDR RNA nanoparticles (right). (B) Same as A but GEM. M: monomer, D: dimer, T: trimer (A and B).

Fig. 2.

Schematics of conjugating PTX onto RNA strand via the click chemistry (top) and incorporating GEM into RNA strands via solid-phase synthesis (bottom).

Fig. 3

(A) Verifying the quality of each 4WJ-FUDR strands and (B) the annealing profile of 4WJ, 4WJ-12-FUDR, 4WJ-22-FUDR, and 4WJ-42-FUDR

Fig. 4

Melting profile of 4WJ, 4WJ-12-FUDR, 4WJ-22-FUDR, and 4WJ-42-FUDR demonstrated by TGGE gel.

Fig. 5. In vitro cytotoxicity of 4WJ-FUDR, 4WJ-GEM, and 4WJ-FUDR-GEM RNA nanoparticles.

(A) Schematics of the construction (left) and cytotoxicity (right) of 4WJ RNA nanoparticles with 0, 22, and 42 copies of FUDR. (B) Schematics of the construction (left) and cytotoxicity (right) of 4WJ RNA nanoparticles with 0, 19, and 42 copies of GEM. (C) Schematics of the construction (left) and cytotoxicity (right) of 4WJ RNA nanoparticles with 31 copies of FUDR and 14 copies of GEM. (D) Schematics of the construction (left) and cytotoxicity (right) of 4WJ RNA nanoparticles with 25 copies of FUDR and 24 copies of GEM. Statistics were calculated by two-tailed unpaired t-test presented as mean ± SD

Fig. 6. Combinational chemotherapy of GEM and PTX by 4WJ RNA nanoparticles.

(A) Schematics of 4WJ RNA nanoparticles with 18 copies of PTX and 6 copies of GEM (top); Cytotoxicity of 4WJ-GEM-PTX with 1 : 3 ratio of GEM : PTX.(bottom). (B) Schematics of 4WJ RNA nanoparticles with 9 copies of GEM (top); Cytotoxicity of 1 : 2 ratio of GEM : PTX (bottom). (C) Dose response matrix of 4WJ-GEM and PTX. (D) HSA synergy map of 4WJ-GEM and PTX (HSA score: 24.956).

Fig. 7. In vivo breast cancer PDX model inhibition of 4WJ-FUDR and 4WJ-GEM.

(A) Mouse weight changes (left), In vivo tumor volume changes (middle), and ex vivo tumor sizes (right) between PBS, FUDR, and 4WJ-FUDR RNA nanoparticles groups (n = 5 biologically independent animals). (B) Mouse weight changes (left), In vivo tumor volume changes (middle) and ex vivo tumor sizes (right panel) between the groups of PBS, GEM, and 4WJ-GEM RNA nanoparticles (n = 5 biologically independent animals). Statistics were calculated by two-tailed unpaired t-test presented as mean ± SEM, *p < 0.05, **p < 0.01. The PBS group is shared between Fig.7A right panel and Fig.7B right panel.

Fig 8.

Ki-67 measurements between PBS, GEM, and 4WJ-GEM RNA nanoparticles groups. Statistics were calculated by two-tailed unpaired t-test presented as mean ± SD, **p < 0.01, ***p < 0.001

Fig. 9. In vivo organ of the safety profile of 4WJ-GEM and 4WJ-FUDR RNA nanoparticles.

Organ weights study comparing PBS, GEM free drug, 4WJ-GEM RNA nanoparticles, FUDR free drug, and 4WJ-FUDR RNA nanoparticles. Statistics were calculated by two-tailed unpaired t-test presented as mean ± SD, *p < 0.05, **p < 0.01, ***P < 0.001.

Fig. 10. In vivo serum biochemistry level study of the safety profile of 4WJ-GEM and 4WJ-FUDR RNA nanoparticles.

Serum biochemistry study comparing PBS, GEM free drug, 4WJ-GEM RNA nanoparticles, FUDR free drug, and 4WJ-FUDR RNA nanoparticles. Alb: albumin; Glo: globulin; TP: total protein; GOT: glutamic oxaloacetic transaminase; GPT: glutamic pyruvic transaminase; CREA: creatinine; BUN: blood urea nitrogen; UA: uric acid; LDH: Lactate dehydrogenase; ALK-P: alkaline phosphatase. Statistics were calculated by two-tailed unpaired t-test presented as mean ± SD, *p < 0.05, **p < 0.01, ***P < 0.001.

Fig. 11. In vivo hematological level study of the safety profile of 4WJ-GEM and 4WJ-FUDR RNA nanoparticles.

Hematological study comparing PBS, GEM free drug, 4WJ-GEM RNA nanoparticles, FUDR free drug, and 4WJ-FUDR RNA nanoparticles. RBC: red blood cell; HGB: hemoglobin; HCT: hematocrit; MCV: mean corpuscular volume; MCH: mean corpuscular hemoglobin; WBC white blood cell; NEUT: neutrophil; MONO: monocyte; EO: eosinophil; BASO: basophil; LYMPH: lymphocyte; PLT: platelet. Statistics were calculated by two-tailed unpaired t-test presented as mean ± SD. *p < 0.05, **p < 0.01, ***P < 0.001.