(T1030-02-08) Adjuvanted Ionizable Lipid Nanoparticles for mRNA Delivery

Purpose: Adjuvants help boost the immunological response against antigens as they act as agonists of pattern-recognition receptors (PRRs) found either on the cell membrane or in subcellular compartments of antigen-presenting cells (APCs) [1]. The most studied adjuvant targets among the PRRs are toll-like receptors (TLRs); however, it is still challenging to stimulate TLRs that are found in subcellular compartments of the APCs. TLR7, an endosomal immune receptor capable of identifying single-stranded RNA, which activates and produces type I interferon and other pro-inflammatory cytokines and promotes an enhanced immune response against viral pathogens [2]. Adifectin (CL347), a lipid-based TLR7 adjuvant, has been shown to efficiently activate TLR7 [3]. The precise intracellular delivery of such TLR7 adjuvants in the endosomal compartment could enhance their adjuvant activity. Ionizable lipid nanoparticles (LNPs) are proven to be versatile and efficient in delivering mRNA into immune cells. The COVID-19 vaccine developed by Moderna and BioNTech Pfizer is a testament to the efficacy of ionizable lipid nanoparticles. Ionizable LNPs possess a neutral charge at physiological pH but get protonated and become positively charged at low pH environments, such as in endolysosomes. This results in membrane instability of the endolysosome and promotes the release of payloads from LNPs. We envision that ionizable LNP technology would synchronously deliver CL347 adjuvant and mRNA precisely to immune cells, allowing enhanced antigen-specific immune responses. Herein, we incorporated CL347 into ionizable LNPs containing the ionizable lipid SM-102 and optimized them for delivering mRNA. CL347 adjuvanted ionizable LNPs were further evaluated for their intracellular delivery and immune cell activation abilities.

Methods: The flash nanoprecipitation (FNP) technique was used to fabricate ionizable LNPs. CL347, the ionizable lipid SM102, and other helper and structural lipid components, including Distearoylphosphatidylcholine (DSPC), 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000 (DMG-PEG2000), and cholesterol, were dissolved in an organic phase containing ethanol. On the other hand, mCherry mRNA was dissolved in an aqueous phase containing sodium citrate buffer (pH 4). The organic and aqueous phases were then impinged against each other in a confined impingement (CIJ) mixer to form adjuvanted LNPs. Unbound mRNA and ethanol were removed from the formulation using a 10 kDa dialysis membrane at room temperature. The particle size and the surface charge of the adjuvanted LNPs were measured using dynamic light scattering (DLS) and electrophoretic light scattering (ELS), respectively. The morphology of the LNPs was confirmed using Transmission Electron Microscopy (TEM). Intracellular delivery of mCherry mRNA from LNPs was verified by confocal live-cell imaging using Raw 264.7 macrophages. Finally, Raw-Blue^TM cells, which are derived from murine RAW 264.7 macrophages and have chromosomal integration of a Secreted embryonic alkaline phosphatase (SEAP) reporter construct inducible by NF-κB and AP-1, were used to assess immune cell activation of the adjuvanted LNPs.

Results: CL347 adjuvanted ionizable LNPs fabricated using the FNP technique were in the size range of 79 to 101 nm with a neutral surface charge at physiological pH (7.4). TEM images confirm the spherical morphology of LNPs (Fig.1A). Encapsulation of mRNA with and without the ionizable lipid SM-102 into LNPs was found to be greater than 90% (Fig.1B). Confocal images show that CL347 ionizable LNPs were able to encode red fluorescent mCherry protein, indicating successful intracellular delivery of mCherry mRNA, whereas CL347 LNPs without an ionizable lipid do not show any mCherry protein encoding inside cells (Fig.2). Interestingly, CL347 ionizable LNPs showed greater immune cell activation, as demonstrated by their significantly higher SEAP activity as compared to free CL347 (Fig.3).

Conclusion: Ionizable LNPs were able to incorporate both mRNA and CL347 without compromising their structural integrity. Adjuvanted ionizable LNPs not only activated immune cells but also successfully processed mRNA inside the cell. Taken together, ionizable LNPs stand out as an effective nanocarrier for the development of adjuvanted mRNA vaccines.

References: 1. Pulendran, B., P. S. Arunachalam, and D.T. O’Hagan, Emerging concepts in the science of vaccine adjuvants. Nature Reviews Drug Discovery, 2021. 20(6): p. 454-475.
2. Petes, C., N. Odoardi, and K. Gee, The Toll for Trafficking: Toll-Like Receptor 7 Delivery to the Endosome. Frontiers in Immunology, 2017. 8.
3. Bixenmann, L., J. Stickdorn, and L. Nuhn, Amphiphilic poly(esteracetal)s as dual pH- and enzyme-responsive micellar immunodrug delivery systems. Polymer Chemistry, 2020. 11(13): p. 2441-2456.

Fig. 1. Physico-chemical characterization of adjuvanted LNPs. A) Particle size of the LNPs measured using dynamic light scattering and number average is reported as d.nm. Negative stained TEM images of CL347-SM102 and CL347 only are reported along with it. The scale is 100 nm. B) Encapsulation efficiency of mCherry mRNA of the CL347 LNPs and CL347-SM102 LNPs measured by QuantiFluor® RNA System. Data represented as mean ± SD (n=4).


Fig. 2. Intracellular localization of mCherry mRNA after 24 hours of incubation of LNPs using Raw 264.7 cells. mCherry mRNA-loaded LNPs successfully encoded into mCherry protein (red color) A) CL347 only mCherry mRNA LNPs were unsuccessful to transfect the mRNA. B) CL347 with 50 molar percent of SM102 is able to efficiently transfect the cells. Live-cell images were taken using a confocal microscope with a 60X objective. The scale bar is 10 μm.

Fig. 3. Secreted embryonic alkaline phosphatase (SEAP) activity of CL347 LNPs after 48 hours of treatment with Raw-Blue™ Cells. Y-axis indicates absorption intensity at 640 nm, directly proportional to the SEAP activity of the samples containing CL347 in LNPs. Data represented as mean ± SD (n=3). Significant differences between each timepoint were determined by one way ANOVA with Tukey’s multiple comparison test, ****p < 0.0006.