(M1230-01-02) Ionizable Lipid Nanoparticles for Precise Intracellular Delivery of CpG DNA Adjuvant

Purpose: Adjuvants act as agonists for pattern-recognition receptors (PRRs), which are located on the cell membrane and in different subcellular compartments such as endosomes, cytosol, and endoplasmic reticulum of antigen-presenting cells (APCs) [1].  Among these PRRs, toll-like receptors (TLRs) are the most researched adjuvant targets; however, stimulation of TLRs located in the intracellular compartments remains a challenge. Synthetic oligodeoxynucleotides (ODNs) containing unmethylated CpG motifs bind to TLR9 within endolysosomes, which promotes the production of proinflammatory cytokines and induces strong cellular and humoral immune responses. CpG DNA is widely used as a TLR9 adjuvant; however, its high hydrophilic nature and poor in vivo stability limit its usage in vaccine formulations [2]. Lipid-based formulations such as liposomes and lipid nanoparticles (LNPs) have been extensively studied as vaccine delivery platforms because of their versatility in accommodating vaccine components and inherent immunostimulatory properties [3]. Specifically, ionizable LNPs are of great interest in the field due to their ability to not only efficiently encapsulate nucleic acid vaccines but also precisely deliver them to intracellular compartments. Of note, ionizable LNP technology was adopted by Moderna and BioNTech-Pfizer to deliver mRNA-based COVID vaccines. Ionizable LNPs stay neutral at physiological pH but are protonated and become positively charged at low pH conditions such as in endolysosomes, which causes membrane instability and enhances the release of payloads from LNPs. We envision that ionizable LNP technology would be helpful in enhancing the encapsulation of negatively charged, hydrophilic CpG DNA and allowing it to be precisely released in low pH endolysosomes. Herein, we prepared ionizable LNPs containing two ionizable lipids, SM-102 and ALC-0315, and optimized them for CpG DNA delivery.

Methods: The flash nanoprecipitation (FNP) technique was used to fabricate ionizable LNPs. Briefly, CpG DNA was dissolved in an aqueous phase containing sodium citrate buffer (pH 4); an organic phase containing ethanol was used to dissolve the ionizable lipids (SM-102 and ALC-0315) at a 10% molar ratio, and other helper and structural lipid components, including DSPC, PEG lipid, and cholesterol. The organic phase is then impinged against an aqueous phase in a confined impingement mixer to rapidly form LNPs. Unbound CpG DNA and ethanol were removed from the formulation using a 10 kDa dialysis membrane at room temperature. The size and surface charge of lipid nanoparticles were measured using dynamic light scattering (DLS) and electrophoretic light scattering (ELS), respectively. The morphology of nanoparticles was confirmed using Transmission Electron Microscopy (TEM). Cellular uptake and intracellular delivery of LNPs were verified in RAW 264.7 macrophages using flow cytometry and confocal live-cell imaging, respectively. For these studies, LNPs were loaded with DiD dye or FITC-conjugated CpG DNA, and lysosomes were stained with Lysotracker^ä dye. Finally, immune cell activation of CpG-loaded LNPs was evaluated using a secreted embryonic alkaline phosphatase (SEAP) reporter assay in Raw-Blue™ Cells, which are derived from the murine RAW 264.7 macrophages with chromosomal integration of a SEAP reporter construct inducible by NF-κB and AP-1.

Results: Ionizable LNPs fabricated using the FNP technique were in the size range of 80 to 100 nm With a neutral surface charge at physiological pH (7.4) (Fig.1). TEM images confirm the spherical morphology of LNPs (Fig.1). Encapsulation of CpG DNA in SM 102 and ALC 0315 LNPs was found to be 64.03 ± 17.54% and 54.26 ± 14.28 %, respectively. Both SM-102 and ALC-0315 LNPs were found to be non-toxic to cells and showed a time-dependent cellular uptake in RAW 264.7 Macrophages. Confocal images clearly showed punctae of DiD dye with Lysotracker^ä dye inside the cells indicating successful localization of LNPs with lysosomes (Fig.2). This was further confirmed with LNPs encapsulated with FITC-conjugated CpG DNA. Interestingly, CpG DNA-LNPs showed comparable SEAP activity to free CpG DNA (Fig. 3), which indicates the release of CpG DNA from ionizable LNPs inside the endosomes to activate the TLR9 receptor.

Conclusion: Flash nanoprecipitation permits the fabrication of ionizable LNPs with high monodispersity. Both SM-102 and ALC-0315 facilitated the higher encapsulation of CpG DNA into LNPs. Ionizable LNPs were effectively taken up by immune cells and precisely delivered CpG DNA inside endosomes. Taken together, ionizable LNPs stand out as an effective nanocarrier to deliver CpG DNA adjuvant

References: Current Opinion in Immunology, 1997. 9(1): p. 4-9.
2. Bode, C., et al., CpG DNA as a vaccine adjuvant. Expert Rev Vaccines, 2011. 10(4): p. 499-511.
3. Huang, W.-C., et al., A malaria vaccine adjuvant based on recombinant antigen binding to liposomes. Nature Nanotechnology, 2018. 13(12): p. 1174-1181.

Acknowledgments: The authors thank LIPOID GmbH for providing the lipids.

Fig. 1. Physical characteristics of LNPs containing ionizable lipids (SM102 (A) and ALC0315 (B)) at a 10% molar ratio. The particle size of the LNPs was measured using dynamic light scattering and is reported as d.nm. Positive stained TEM images of SM102 and ALC0315 LNPs are reported along with it.


Fig. 2. Intracellular co-localization of DiD-loaded LNPs in RAW 264.7 cells. A merged confocal image was obtained after 24 hours of incubation of LNPs with RAW 264.7 cells. Live-cell images were taken using a confocal microscope with a 100X objective and punctae inside the cells indicated by white arrows.


Fig. 3. Secreted embryonic alkaline phosphatase (SEAP) activity of CpG DNA-loaded LNPs and Free CpG after 48 hours of treatment with Raw-Blue™ Cells. Y-axis indicates absorption intensity at 640 nm that is directly proportional to the SEAP activity of the samples containing different concentrations of CpG DNA encapsulated in LNPs or Free CpG DNA. FBS and Lipopolysaccharide (LPS) were used as negative and positive controls, respectively.