In Vitro and In Vivo Evaluation of the Pathology and Safety Aspects of Three- and Four-Way Junction RNA Nanoparticles

Abstract

RNA therapeutics has advanced into the third milestone in pharmaceutical drug development, following chemical and protein therapeutics. RNA itself can serve as therapeutics, carriers, regulators, or substrates in drug development. Due to RNA's motile, dynamic, and deformable properties, RNA nanoparticles have demonstrated spontaneous targeting and accumulation in cancer vasculature and fast excretion through the kidney glomerulus to urine to prevent possible interactions with healthy organs. Furthermore, the negatively charged phosphate backbone of RNA results in general repulsion from negatively charged lipid cell membranes for further avoidance of vital organs. Thus, RNA nanoparticles can spontaneously enrich tumor vasculature and efficiently enter tumor cells via specific targeting, while those not entering the tumor tissue will clear from the body quickly. These favorable parameters have led to the expectation that RNA has low or little toxicity. RNA nanoparticles have been well characterized for their anticancer efficacy; however, little detail on RNA nanoparticle pathology and safety is known. Here, we report the in vitro and in vivo assessment of the pathology and safety aspects of different RNA nanoparticles including RNA three-way junction (3WJ) harboring 2′-F modified pyrimidine, folic acid, and Survivin siRNA, as well as the RNA four-way junction (4WJ) harboring 2′-F modified pyrimidine and 24 copies of SN38. Both animal models and patient serum were investigated. In vitro studies include hemolysis, platelet aggregation, complement activation, plasma coagulation, and interferon induction. In vivo studies include hematoxylin and eosin (H&E) staining, hematological and biochemical analysis as the serum profiling, and animal organ weight study. No significant toxicity, side effect, or immune responses were detected during the extensive safety evaluations of RNA nanoparticles. These results further complement previous cancer inhibition studies and demonstrate RNA nanoparticles as an effective and safe drug delivery vehicle for future clinical translations.

Cracking the Code: Enhancing Molecular Tools for Progress in Nanobiotechnology

“…Subsequent evaluations showcased the nanoparticles’ effectiveness in inhibiting primary tumors as well as lung metastasis of colorectal cancer. The design, quality assessment, and safety evaluation of RNA nanoparticles intended for drug delivery were also evaluated . Two types of RNA nanoparticles were initially designed: a 3WJ scaffold with specific modifications and a 2′F 3WJ scaffold incorporating siRNA targeting survivin.…”

“…Human genome sequencing has revealed that a majority of nonprotein-coding DNA encodes for noncoding RNAs, marking a paradigm shift that could indicate RNA therapeutics as the third great milestone in pharmaceutical drug development. , The inherent motility and deformability of RNA nanoparticles allow for swift and efficient tumor accumulation through both spontaneous and active targeting, while their negative charge and dynamic nature enable rapid renal excretion for nontumor-accumulated RNA nanoparticles, minimizing potential toxicity concerns. , Additionally, the incorporation of ligands for cancer targeting has further enhanced RNA nanoparticle biodistribution. With properties of self-assembly, programmability, and multivalency, RNA nanoparticles present as a promising material for pharmaceutical applications. − These studies explored the application of RNA–drug conjugation to facilitate the controlled release of drugs into their original forms and the utilization of arrow tail RNA for targeted drug delivery to cancer cell cytosols via exosome surface display. , Remarkably, both RNA nanoparticles and RNA-displaying exosomes demonstrated potent anticancer capability, inhibiting cancers and their metastasis efficiently without discernible toxicity. ,,,− …”

“…The design, quality assessment, and safety evaluation of RNA nanoparticles intended for drug delivery were also evaluated. 28 Two types of RNA nanoparticles were initially designed: a 3WJ scaffold with specific modifications and a 2′F 3WJ scaffold incorporating siRNA targeting survivin. Quality checks were conducted to confirm the absence of bacterial contamination and ensure low endotoxin levels.…”

“… 57 , 58 Remarkably, both RNA nanoparticles and RNA-displaying exosomes demonstrated potent anticancer capability, inhibiting cancers and their metastasis efficiently without discernible toxicity. 27 , 28 , 57 , 59 − 62 …”