(W1030-07-37) Production and Optimization of Nanosac as a Soft Nanocarrier for 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 four 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 organs beyond the liver, without significant toxicities. To this end, we have developed Nanosac, a soft and non-cationic gene carrier to enable systemic delivery of short-interfering RNA (siRNA) suppressing immune checkpoint interactions for solid tumor therapy [3]. We selected PD-1/PD-L1 and CD47/SIRPa as target immune checkpoints due to their critical roles as “don't-find-me” and “don't-eat-me” signals to immune cells, which interfere with the development of adaptive and innate antitumor immunity, respectively.

Methods: Nanosac was produced using a sacrificial template of mesoporous silica nanoparticles (MSN), which were surface-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 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.

Results: The Nanosac preparation was optimized by varying the following parameters: time and pH of pD coating, strength of the fluoride buffer used to dissolve the silica template, and the siRNA loading capacity. Increasing the pD coating time from 6 to 24 h increased the pD layer thickness from 15 nm to 35 nm, which contributed to supporting the hollow Nanosac structure after silica core removal (Fig. 1a). We also observed that increasing the pH of pD coating from 7.9 to 8.5 formed more uniform and continuous pD layer, which helped maintain the NP integrity during the subsequent steps (Fig. 1b). Etching silica using [F⁻] of 10 M resulted in significant distortion of the protective pD layer, whereas [F⁻] of 0.2 M was insufficient and showed very little change of silica structure (Fig. 1c). [F⁻] of 1.8 M achieved selective removal of silica core without affecting the pD coating. siRNA loading could be increased to 5 wt%, in contrast to the current LNPs formulations with a typical siRNA loading of ~1 wt% [4]. Nanosac prepared at different siRNA loading ratios (0.7-5.4 wt%) silenced a reporter gene (luciferase) to similar extents (Fig. 1d). Nanosac loaded with siRNAs targeting PD-L1 or CD47 (siPD-L1 or siCD47) achieved gene knockdown efficiency comparable to Lipofectamine 2000^®, a cationic transfection agent serving as a commercial benchmark, in B16F10 melanoma cells (Fig. 2a and 2b). Further, Nanosac lyophilized with trehalose maintained its physical stability and biological activity (Fig. 2c), indicating the potential for cold-chain free storage and transportation.

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 preparation parameters were optimized to improve the final product quality. The biodistribution and anti-tumor efficacy of siRNA-loaded Nanosac in tumor-bearing mice are 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.
4. Da Silva Sanchez, A.J., et al., Universal Barcoding Predicts In Vivo ApoE-Independent Lipid Nanoparticle Delivery. Nano Letters, 2022.

Acknowledgments: We acknowledge the financial support from NIH R01 CA258737, Purdue Center for Cancer Research, and Purdue Institute for Drug Discovery. The authors report no conflict of interest.

Fig. 1. Optimization Nanosac preparation. (a) Effect of polydopamine (pD) coating time, (b) Effect of pD coating pH, (c) Effect of the fluoride buffer strength, and (d) Effect of siRNA loading wt% on the Nanosac properties.



Fig. 2. Gene knockdown efficiency of Nanosac. (a) PD-L1, (b) CD47, and (c) PD-L1 gene knockdown efficiency of lyophilized Nanosac.