(M1530-01-05) Nanosac: A Soft Nanocarrier for Systemic siRNA Delivery to Solid Tumors

Purpose: Nucleic acids have gained a significant interest in drug delivery community owing to their potential to address diseases with few “druggable” targets. Nevertheless, nucleic acids are challenging drug candidates from the delivery standpoint due to their large molecular weights, dense negative charges, pro-inflammatory activities, and short half-lives in biological fluids. Synthetic gene carriers have been explored; yet, their cationic nature results in significant toxicities and accelerated removal by the filtering organs upon systemic administration [1] . In the past five years, a growing number of nucleic acid-based therapeutics have been approved by the FDA, formulated as ionizable lipid nanoparticles (LNPs) for the treatment of hepatic disorders. However, LNPs show > 80% accumulation in the liver after systemic administration [2]. Therefore, there is a critical unmet need for a nanocarrier capable of encapsulating and protecting nucleic acids and enabling their systemic delivery to the organs beyond the liver, without significant toxicities. To this end, we have developed Nanosac, a soft and non-cationic nanocarrier to enable systemic delivery of short-interfering RNA (siRNA) suppressing immune checkpoint interactions for solid tumor therapy [3]. We selected CD47/SIRPa and PD-l/PD-L1 as target immune checkpoints due to their critical roles as “don't-eat-me” and “don't-find-me” signals to immune cells, which interfere with the development of innate and adaptive antitumor immunity, respectively.

Methods: Nanosac was produced using a sacrificial template of mesoporous silica nanoparticles functionalized with cationic amine groups (MSN^a) to complex with anionic siRNA. MSN^a was coated with a protective polydopamine (pD) layer to entrap and shield the siRNA from nucleases. In the final step, the MSN^a template was selectively etched out with fluoride buffer to produce hollow Nanosac. Lyophilized Nanosac was produced by mixing Nanosac with trehalose as a cryoprotectant in 1:2 w/w ratio. Gene knockdown efficiency of fresh or lyophilized Nanosac was tested in vitro in murine B16F10 melanoma cells. The antitumor efficacy of siRNA-loaded Nanosac was evaluated in subcutaneous B16F10 melanoma and CT26 colon carcinoma mouse models and compared to LNPs. The treatments were administered intravenously at each siRNA dose of 0.75 mg/kg every 4 days with a total of 3 or 5 doses, depending on the tumor model.

Results: 1. Nanosac optimization and in vitro characterization: The Nanosac preparation was optimized by varying the preparation parameters. Increasing the pD coating time from 6 to 24 h or the pH of pD coating from 7.9 to 8.5 increased the pD layer thickness, which better supported the hollow Nanosac structure after core removal (Fig. 1a and b). [F⁻] of 1.8 M was optimal for selective removal of silica core. Higher [F⁻] (10 M) destroyed the pD layer, and lower [F⁻] (0.2 M) was insufficient (Fig. 1c). Nanosac loaded with siRNA targeting PD-L1 or CD47 (siPD-L1 or siCD47) achieved gene knockdown efficiency comparable to Lipofectamine 2000^®, a cationic transfection agent, in B16F10 melanoma cells (Fig. 1d) without reducing cell viability at concentrations up to 1 mg/mL. Further, Nanosac lyophilized with trehalose maintained its physical stability and biological activity, indicating the potential for cold-chain free storage and transportation. 2. In vivo antitumor efficacy: First, we evaluated the antitumor efficacy of the optimized Nanosac in subcutaneous B16F10 melanoma-bearing mice representing an immune-cold tumor model. Tumors of the negative control group (D5W) grew aggressively with a median survival time of 12 days (Fig. 2a). Nanosac delivering either siCD47 or siPDL1 suppressed the tumor growth rate slightly. In contrast, the mixture of two siRNA-loaded Nanosacs suppressed the tumor growth best, extending the median survival time to 26 days (Fig. 2a and c). Notably, with the same set of siRNAs, the Nanosac achieved greater antitumor effect than LNPs containing the ionizable lipid SM-102 (Fig. 2a and b), analogous to the LNPs used in the Moderna Covid-19 mRNA vaccine (Spikevax®). An immune-hot tumor model of CT26 colon carcinoma showed similar result as the B16F10 model. Nanosac suppressed the tumor growth to a greater extent than the LNPs (Fig. 2d) and significantly prolonged the mouse survival, with 25% of mice showing complete tumor regression (Fig. 2e).

Conclusion: Nanosac has been developed and optimized as a promising non-cationic and versatile gene carrier to fill the current gap in systemic delivery of nucleic acid therapeutics. The optimized Nanosac silenced the target immune checkpoint genes in tumor cells in vitro and showed greater tumor suppression and survival rate than the LNPs with SM-102 in two different tumor-bearing mouse models. The Nanosac biodistribution is currently under investigation.

References: 1. Ogris, M., et al., PEGylated DNA/transferrin–PEI complexes: reduced interaction with blood components, extended circulation in blood and potential for systemic gene delivery. Gene Therapy, 1999. 6(4): p. 595-605.
2. Kulkarni, J.A., et al., The current landscape of nucleic acid therapeutics. Nature Nanotechnology, 2021. 16(6): p. 630-643.
3. Kim, H., et al., Nanosac, a Noncationic and Soft Polyphenol Nanocapsule, Enables Systemic Delivery of siRNA to Solid Tumors. ACS nano, 2021. 15(3): p. 4576-4593.

Acknowledgements: We acknowledge the financial support from NIH R01 CA258737, Purdue Center for Cancer Research, and Purdue Institute for Drug Discovery.

Fig. 1. Optimization and in vitro characterization of Nanosac. The effect of polydopamine (pD) coating time (a), pD coating pH (b), Fluoride buffer strength (c) on Nanosac properties. Gene knockdown efficiency of Nanosac loaded with siPD-L1 or siCD47 (d).

Fig. 2. In vivo antitumor efficacy of siRNA-loaded Nanosac in B16F10 melanoma (a-c) or CT26 colon carcinoma tumor model (d-e). Individual tumor growth curves (a, d), Tumor specific growth rate (b), and Kaplan-Meier survival curves (c, e). Red arrows indicate IV dosing. Numbers in parenthesis indicate the median survival time (in days).