(T1330-03-16) pRNA Nanoparticles for Ocular Delivery: Pharmacokinetics and Pharmacodynamics After Subconjunctival Injection

Purpose: Choroidal neovascularization is one of the causes leading to irreversible vision loss and is associated with various posterior eye segment diseases such as diabetic retinopathy and wet age-related macular degeneration ¹. RNA-based therapeutic agents including small interfering RNA, RNA aptamers, and miRNA are promising in disease treatment ². However, RNA molecules are unstable because of degradation in the body ^{3, 4}. Recently, it was discovered that the RNA three-way junction motif (3WJ) from the packaging RNA (pRNA) of bacteriophage phi29 DNA packaging motor can fold into a compact structure and can constitute an RNA nanoparticle, which can combine with multiple function modules as a nanodelivery system (pRNA nanoparticles) ^{5, 6}. These systems were shown to be thermodynamically and chemically stable ^{5, 6}. The purposes of this project were to evaluate the pRNA nanoparticles for ocular delivery and to demonstrate that pRNA nanoparticles could deliver the relevant therapeutic reagents to treat choroidal neovascularization in the posterior eye segment.

Methods: The pRNA nanoparticles evaluated in this study were (a) “backbone” itself only as the carrier system with different sizes: small size nanoparticles pRNA-3WJ, larger nanoparticles pRNA-Sqaure (SQR), RNA-4WJ, RNA-6WJ, and RNA-8WJ; and (b) nanoparticles containing therapeutic aptamers (single or multiple anti-vascular endothelial growth factor (VEGF) aptamers or anti-VEGF and anti-angiopoietin-2 (Ang2)) chemically linked to the “backbone” (e.g., SQR-VEGF). The pharmacokinetics of pRNA nanoparticles was evaluated in a mouse model. First, the clearance of pRNA nanoparticles in the eye was monitored by whole-body fluorescence imaging at predetermined timepoints after 2.5 µM, 10 µl subconjunctival injection of pRNA nanoparticles in vivo. The images were analyzed, and the mean fluorescence intensity of the eye region was determined. Second, the distribution and delivery of pRNA nanoparticles in eye tissues were demonstrated by confocal microscopy of dissected eye tissues including cornea, retina, and sclera at 6 h and 24 h after subconjunctival injection of pRNA nanoparticles. The pharmacodynamics of pRNA nanoparticles were evaluated by WST-1 cell proliferation assay on human umbilical vein endothelial cells (HUVEC) as the model. Four-six thousand cells were seeded in 96-well plate and incubated for 48 h to allow attachment. The cells were then treated with the medium containing different concentrations of pRNA nanoparticles for 48 h and then incubated with WST-1 reagent to compare the antiangiogenic effects of the different pRNA nanoparticles.

Results: The results of the whole-body imaging experiments showed the clearance profiles of the pRNA nanoparticles (Fig. 1). The small size nanoparticles, pRNA-3WJ nanoparticles except for 3WJ-3VEGF, were cleared from the eye within 5 hours, which was similar to the clearance of the fluorescent dye only (p > 0.05). The nanoparticles with larger sizes including pRNA-Square, 4WJ, 6WJ, and 8WJ had longer clearance up to 48 h compared to the smaller nanoparticles. In addition, attaching aptamers to nanoparticle “backbone” in all types of nanoparticle systems did not affect the clearance profile of the nanoparticles at all timepoints (p > 0.05). In the second part of the study using confocal microscopy, the images of dissected tissues treated with SQR-VEGF demonstrated the delivery of nanoparticles at the cellular level. In the WST-1 proliferation assay, the results showed the antiangiogenic effects of nanoparticles with the aptamers (Fig. 2). Particularly, the effects were more pronounced starting at 0.1 µM of all nanoparticles. There was no significant difference between 0.4 µM and 0.1 µM of single aptamer nanoparticles SQR-VEGF and 8WJ-VEGF. However, nanoparticles with multiple aptamers had significantly higher efficacy than that of nanoparticles with single aptamer at both 0.1 µM and 0.4 µM (p < 0.01 for 3WJ-3VEGF vs. 3WJ-1VEGF, p < 0.0001 for SQR-VEGF-Ang2 vs. SQR-VEGF at both concentrations).

Conclusion: The rates of clearance from the eye decreased when the sizes of the pRNA nanoparticle increased and the conjugation of the aptamers to the nanoparticle “backbone” did not affect its clearance and PK profile. The pRNA nanoparticles were demonstrated to be delivered to the posterior eye segment at the cellular level. Additionally, the WST-1 proliferation assay indicated that the pRNA nanoparticles had desired antiangiogenic effects.

References: 1. Yeo, N. J. Y.; Chan, E. J. J.; Cheung, C., Choroidal Neovascularization: Mechanisms of Endothelial Dysfunction. Front Pharmacol 2019, 10, 1363.
2. Fattal, E.; Bochot, A., Ocular delivery of nucleic acids: antisense oligonucleotides, aptamers and siRNA. Advanced Drug Delivery Reviews 2006, 58 (11), 1203-1223.
3. Roberts, T. C.; Langer, R.; Wood, M. J. A., Advances in oligonucleotide drug delivery. Nature Reviews Drug Discovery 2020, 19 (10), 673-694.
4. Burnett, J. C.; Rossi, J. J., RNA-based therapeutics: current progress and future prospects. Chem Biol 2012, 19 (1), 60-71.
5. Shu, D.; Shu, Y.; Haque, F.; Abdelmawla, S.; Guo, P., Thermodynamically stable RNA three-way junction for constructing multifunctional nanoparticles for delivery of therapeutics. Nature Nanotechnology 2011, 6 (10), 658-667.
6. Guo, S.; Piao, X.; Li, H.; Guo, P., Methods for construction and characterization of simple or special multifunctional RNA nanoparticles based on the 3WJ of phi29 DNA packaging motor. Methods 2018, 143, 121-133.

Acknowledgements: The authors would like to thank Dr. Winston W-Y. Kao, Dr. Chia-yang Liu, Dr. Yong Yuan, and Jianhua Zhang for their suggestions and help; Dr. Lisa Lemen and Xiangning Wang at the Vontz Core Imaging Laboratory, University of Cincinnati, for their help in the whole-body imaging study; Dr. Bingfang Yan for providing the cell culture facilities; Mohammed Hassan for his help in this project. This research was supported by National Institutes of Health (NIH) grant R01EY031452.

Fig. 1. % Mean fluorescence intensity based on the mean intensity at t = 0 versus time after subconjunctival injection in mouse eye in vivo. Mean ± SD (n = 3-5).

Fig. 2. (a) Percent effect based on positive control 0.1 µM bevacizumab (Avastin) as 100% effect versus different concentration levels of pRNA nanoparticles on HUVEC model; comparison of percent effect based on positive control among (b) pRNA-3WJ nanoparticles, (c) pRNA-square (SQR) nanoparticles, and (d) 8WJ nanoparticles at two higher concentrations: 0.1 and 0.4 µM. Mean ± SEM (n = 12-44, except 0.1 and 2 µM Avastin with n = 179), ns p > 0.05; * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001.