(M1530-04-20) Reducing the Pro-Inflammatory and Cytotoxic Side Effects of siRNA- Solid Lipid Nanoparticles by Optimizing Cationic Lipid; DOTAP

Purpose: Cationic lipids are commonly used to enhance siRNA encapsulation in solid lipid nanoparticles (SLNs). However, they are also primarily responsible for the cytotoxicity and pro-inflammatory activity of the SLNs. Previously, we reported a novel TNF-α siRNA-encapsulated SLNs formulation that showed strong activity against rheumatoid arthritis unresponsive to methotrexate. This study was designed to optimize the cationic lipid (DOTAP) amount in the TNF-α siRNA-SLNs to reduce their pro-inflammatory activity and cytotoxicity.

Methods: DOTAP-siRNA complexes prepared at various Nitrogen to Phosphate charge ratios (N/P, 34:1 to 1:1) were encapsulated in the SLNs. The physical properties of the resultant SLNs were characterized, and their cytotoxicity and ability to induce pro-inflammatory cytokines were evaluated in J774A.1 macrophages in culture. In-vivo pharmacokinetics and biodistribution characterization of the optimized formulation 12:1 was also investigated.

Results: The complexation efficiency of siRNA to DOTAP was reduced from ~100% at N/P ratio 34:1 to ~80% at N/P ratios 12:1 and 1:1. The particle size of the TNF-α siRNA-SLNs was decreased from 141.4 ± 4.5 nm to 81 ± 3 nm and their zeta potential was changed from 5.8 ± 7.2 to - 44.2 ± 3.1 mV as the N/P ratio decreased from 34:1 to 1:1. The burst release of siRNA from all SLNs was minimal. As expected, the siRNA-SLNs prepared at the highest N/P ratio (34:1) showed the highest cytotoxicity to J774A.1 cells, and reducing the N/P ratio reduced the cytotoxicity of the SLNs, except that the SLNs prepared at the N/P ratio of 1:1 were surprisingly more cytotoxic than SLNs prepared at N/P ratios of 12:1 and 20:1. The relatively faster release of DOTAP from the siRNA-SLNs prepared at the N/P ratio of 1:1, as compared to that from the siRNA-SLNs prepared at the N/P ratio of 12:1, was likely responsible to the higher cytotoxicity of the siRNA-SLNs prepared at the P/N ratio of 1:1. The siRNA-SLNs prepared at N/P ratios of 12:1 and 1:1 showed comparable pro-inflammatory activities, but significantly weaker than siRNA-SLNs prepared at the N/P of 34:1. The TNF-α siRNA in the SLNs prepared at N/P ratios of 34:1, 12:1, or 1:1 was equally effective in downregulating TNF- α expression in J774A.1 macrophages. The optimized siRNA-SLNs that were prepared at the N/P ratio of 12:1 showed significant distribution in tumor tissues in a mouse model, with a plasma half-life of 70 h, indicating a prolonged circulation in blood upon intravenous administration.

Conclusion: In conclusion, we have identified a new TNF-α siRNA-SLNs formulation with minimum pro-inflammatory activity and cytotoxicity.

References: (1) Aldayel, A. M.; O'Mary, H. L.; Valdes, S. A.; Li, X.; Thakkar, S. G.; Mustafa, B. E.; Cui, Z., Lipid nanoparticles with minimum burst release of TNF-alpha siRNA show strong activity against rheumatoid arthritis unresponsive to methotrexate. Journal of controlled release: official journal of the Controlled Release Society 2018, 283, 280-289.
(2) Hannah L. O’Mary, Mahmoud S. Hanafy, Abdulaziz M. Aldayel, Solange A. Valdes, Riyad F. Alzhrani, Stephanie Hufnagel, John J. Koleng, Zhengrong Cui. Effect of the Ratio of Betamethasone to TNF-α siRNA Coencapsulated in Solid Lipid Nanoparticles on the Acute Proinflammatory Activity of the Nanoparticles. Mol. Pharmaceutics. (2019).
(3) Lobovkina T, Jacobson GB, Gonzalez-Gonzalez E, Hickerson RP, Leake D, Kaspar RL, Contag CH, Zare RN. In vivo sustained release of siRNA from solid lipid nanoparticles. ACS nano. 2011 Dec 27;5(12):9977-83.

Acknowledgements: This work was supported by the Egyptian Government Scholarship.


Fig.1: Cytotoxicity of different siRNA-SLNs prepared at cationic DOTAP to siRNA ratio (N/P ratio) 34:1 to 1:1 using a standard MTT assay.


Fig.2: Evaluate the proinflammatory activity of siRNA-SLNs by its ability to stimulate IL-6 production by J774A.1 macrophages was determined.


Fig.3: Biodistribution profile differences of formulations 34:1 and the optimized 12:1 N/P ratio (P;Pancrease, Sp;Spleen, K;Kidney, Lv;Liver, H;Heart, L;lungs, O&U;Ovaries and Uterus, B;Brain, S;Stomach, S.int;Small Intestine, L.int;Large Intestine).