(T1130-09-55) Systemic Delivery of siRNA to Solid Tumors with Nanopuff Polyphenol Nanoparticles

Purpose: Small interfering RNA (siRNA), 21-nucleotide double-stranded RNA that silences specific gene translation, has emerged as a potent therapeutic agent. However, siRNA is unstable in a physiological environment and does not enter cells efficiently because of its negative charges. Therefore, an efficient carrier is necessary for developing siRNA therapeutics. Cationic nanoparticles (NPs), lipid nanoparticles (LNPs), and N-acetylgalactosamine (GalNac)-conjugates have been used to improve the stability and cellular uptake of siRNA. However, cationic carriers can cause toxicity or suffer from phagocytic clearance due to opsonization. LNPs and GalNac-conjugates have limitations in reaching extrahepatic organs. Our goal is to develop a non-cationic siRNA carrier that can deliver siRNA efficiently to extrahepatic organs including solid tumors. Toward this goal, we have developed albumin-bathed polydopamine nanoparticles (Nanopuff) that load siRNA by hydrogen boding and protect the siRNA from enzymatic degradation.

Methods: Polydopamine nanoparticles (PDNPs) were synthesized by oxidative self-polymerization of dopamine in alkaline buffer. Particle size, zeta-potential, and catechol-rich surface were confirmed by dynamic light scattering and UV-Vis spectroscopy. We treated PDNPs in 10 µM ascorbic acid solution for 1 h to restore catechol dominant surface, which enabled siRNA loading at neutral pH. We loaded siRNA on the ascorbic acid-treated PDNPs by incubation in nuclease free water for 2 h and bathed the siRNA-loaded PDNP in 5 mg/mL human serum albumin solution for 1 h to coat the surface with albumin. siRNA binding on the surface of PDNPs and the RNase protection were determined by agarose gel electrophoresis. The albumin-coated, siRNA-loaded PDNP were called Nanopuff. We treated B16F10 cells with fluorescently labeled siRNA-loaded Nanopuff (siRNA@Nanopuff) and quantified colocalization of Nanopuff and lysosomal tracker, comparing to Lipofectamine 3000. We evaluated siRNA delivery by Nanopuff in vitro using PD-L1-targeting siRNA (siPD-L1) and B16F10 melanoma cells and quantified PD-L1 mRNA levels in the treated cells by RT-qPCR. For in vivo efficacy, we administered siPD-L1@Nanopuff to CT26 colon carcinoma-bearing mice by intravenous injection and monitored tumor volumes to evaluate therapeutic efficacy.

Results: Surface chemistry and siRNA loading. Agarose gel electrophoresis showed that pretreatment of PDNPs with ascorbic acid increased the fraction of catechol on the surface, enhancing siRNA binding to the NPs at neutral pH without introducing cationic charges. Albumin coating of siRNA@PDNP helped protect siRNA from RNase. Cellular uptake pathway and intracellular trafficking. Cellular uptake of Nanopuff was confirmed by confocal microscopy. Both PDNP and Nanopuff showed minimal colocalization with the lysosomal tracker, whereas the Lipofectamine exhibited strong lysosomal colocalization (Fig. 1). Since clathrin-mediated endocytosis typically evolves lysosomal trafficking, the lack of Nanopuff-lysosome colocalization suggests that Nanopuff enter cells by non-clathrin-mediated endocytosis. Gene silencing efficacy. In B16F10 and CT26 cells, siPD-L1@Nanopuff induced a significant reduction in PD-L1 mRNA level compared to the untreated group. siPD-L1@Nanopuff was comparable to siPD-L1@LNP (MC-3) (Fig. 2a) siPD-L1 @lipofectamine in PD-L1 silencing (Fig. 2b). In vivo efficacy of PD-L1-Nanopuff. In the CT26 colon carcinoma model, systemic administration of siPD-L1@Nanopuff significantly attenuated tumor growth compared to dextrose 5% (D5W) and siPD-L1@LNP (MC-3) (Fig. 3a). No significant changes in body weight were observed after Nanopuff administration, suggesting no systemic toxicity (Fig. 3b).

Conclusion: We developed Nanopuff - non-cationic polyphenol nanoparticles that protect siRNA from enzymatic degradation and deliver the siRNA to silence the target protein. siPD-L1@ Nanopuff silenced PD-L1 expression in B16F10 and CT26 cells. Systemic administration of siPD-L1@Nanopuff suppressed tumor growth better than LNPs in CT25 tumor-bearing mice without noticeable systemic toxicity. Nanopuff is a promising nanocarrier with the potential to deliver siRNA to extrahepatic organs, including solid tumors.

Acknowledgements: This work was supported by the National Institutes of Health (R01CA258737)

Fig. 1. (a) Confocal microscope images locating Cy3-siRNA-loaded Lipofectamine 3000, PDNPs, and Nanopuff relative to lysosomes in B16F10 cells. Green: Lysotracker (lysosome); red: Cy3-labeled siRNA; and blue: DAPI (nuclei). (b) Pearson’s correlation coefficients indicating the degree of siRNA & lysosome colocalization in confocal images: R = 1 (perfect colocalization), and R = 0 (no colocalization). n = 3 confocal dish of a representative image (mean ± SD).

Fig. 2 RT-qPCR analysis of PD-L1 gene silenced by siPD-L1@LNP(MC3), siPD-L1@L3K, siPD-L1@PDNPs, and siPD-L1@Nanopuff (albumin-bathed PDNP) in (a) IFN-gamma activated B16F10 and (b) IFN-gamma activated CT26 cells. n = 3 test of a representative batch, mean ± SD, Tukey's multiple comparisons test following one-way ANOVA.

Fig 3. Antitumor efficacy of D5W, siPD-L1@LNP, and siPD-L1@Nanopuff in Balb/c mice bearing CT26 tumors (siPD-L1: 0.75 mg/kg, Intravenous injected every 3 days for 5 times). n = 5 mice per group (mean ± stdev). Tumor volume (a) and weight of the mice (b) were measured every 3 days to evaluate the efficacy and toxicity of the particles.