(T1130-03-15) Albumin corona Improves Colloidal Stability of Nanosac, the Non-cationic Polyphenol Gene Carrier

Purpose: To avoid the toxicity of cationic carriers and improve extrahepatic delivery of siRNA, we have developed a nanocapsule system called “Nanosac”. Nanosac is a soft, non-cationic nanocapsule encapsulating siRNA inside [1]. Nanosac offers two unique features, negatively charged surface and softness, which are particularly advantageous for systemic gene delivery. Nanosac does not depend on cationic charges to load nucleic acids, thus avoiding toxicity and non-specific protein adsorption. This facilitates intravenous administration of Nanosac with a minimal risk of systemic side effects. Softness of Nanosac is also favorable for evading the reticuloendothelial system uptake [2-5]. With these features, Nanosac delivered siRNA targeting PD-L1 (siPD-L1) to tumors systemically, proving superior to anti-PD-L1 antibody and a hard counterpart in carrying siPD-L1 in tumor suppression [1]. Given the effectiveness of Nanosac in vivo, we speculate that Nanosac is colloidally stable in circulation, a critical feature to its systemic application. However, it remains unclear how Nanosac interacts with components in physiological fluids. The objective of study is to investigate the collodial stability of Nanosac in physiological medium and contributing factors to its colloidal status.

Methods: Nanosac was produced by sequential coating of mesoporous silica nanoparticles (MSN) with siRNA and polydopamine (pD), followed by removal of the sacrificial MSN core by fluoride buffer. Fresh Nanosac samples were suspended in electrolyte solutions (150 mM NaCl, 150 mM KCl or phosphate buffered saline, PBS), albumin solution (40 mg/mL), or a solution of albumin and electrolyte mixture. Alternatively, Nanosac was mixed with albumin first and then with electrolytes. The size of Nanosac in these solutions were measured by dynamic light scattering. For prealbuminylation, Nanosac was incubated in albumin solutions with different albumin concentrations (0.5-80 mg/mL) overnight at room temperature. The prealbuminylated Nanosac was added to PBS, and the size was measured.

Results: The effect of electrolytes on colloidal stability of Nanosac was investigated in various electrolyte solutions (Fig.1a). Nanosac was prone to aggregation in the presence of electrolytes such as NaCl, KCl, or PBS, due to the neutralization of the negative charge of Nanosac. Nanosac did not aggregate in albumin solution (Fig.1b). When albumin was added prior to electrolytes, Nanosac did not aggregate in electrolyte solution. However, when Nanosac was added to the solution of albumin and electrolyte mixture, it underwent aggregation (Fig. 1c). Prealbuminylated Nanosac, especially in relatively concentrated albumin (10-80 mg/mL), was protected from electrolyte-induced aggregation in PBS (Fig. 2).

Conclusion: Preexposure to albumin was critical to the colloidal stability of Nanosac. Given the safety and effectiveness of Nanosac in systemic application [1], Nanosac is likely to have been protected by in situ albuminylation in the abundance of blood proteins before they were subjected to the effect of electrolytes. We are currently investigating how prealbuminylation affects the circulation and cellular uptake of Nanosac.

References: 1. 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.
2. Liang, Q., et al., The softness of tumour-cell-derived microparticles regulates their drug-delivery efficiency. Nature biomedical engineering, 2019. 3(9): p. 729-740.
3. Anselmo, A.C. and S. Mitragotri, Impact of particle elasticity on particle-based drug delivery systems. Advanced drug delivery reviews, 2017. 108: p. 51-67.
4. Anselmo, A.C., et al., Elasticity of nanoparticles influences their blood circulation, phagocytosis, endocytosis, and targeting. ACS nano, 2015. 9(3): p. 3169-3177.
5. Key, J., et al., Soft discoidal polymeric nanoconstructs resist macrophage uptake and enhance vascular targeting in tumors. ACS nano, 2015. 9(12): p. 11628-11641.

Acknowledgements: We acknowledge the financial support from NIH R01 CA258737, Purdue Center for Cancer Research, and Purdue Institute for Drug Discovery. We also acknowledge the Fellowship support from the Egyptian Government Ministry of Higher Education Missions Sector to M.E.

Fig. 1. Nanosac colloidal stability. Effect of (a) electrolyte, (b) albumin, and (c) electrolyte and albumin.

Fig. 2. Effect of pre-albuminylation on colloidal stability of Nanosac in PBS.