Enhancing the Stability and Immunomodulatory Activity of Liposomal Spherical Nucleic Acids through Lipid‐Tail DNA Modifications

Liposomal spherical nucleic acids (LSNAs) are an attractive therapeutic platform for gene regulation and immunomodulation due to their biocompatibility, chemically tunable structures, and ability to enter cells rapidly without the need for ancillary transfection agents. Such structures consist of small (<100 nm) liposomal cores functionalized with a dense, highly oriented nucleic acid shell, both of which are key components in facilitating their biological activity. Here, the properties of LSNAs synthesized using conventional methods, anchoring cholesterol terminated oligonucleotides into a liposomal core, are compared to LSNAs made by directly modifying the surface of a liposomal core containing azide‐functionalized lipids with dibenzocyclooctyl‐terminated oligonucleotides. The surface densities of the oligonucleotides are measured for both types of LSNAs, with the lipid‐modified structures having approximately twice the oligonucleotide surface coverage. The stabilities and cellular uptake properties of these structures are also evaluated. The higher density, lipid‐functionalized structures are markedly more stable than conventional cholesterol‐based structures in the presence of other unmodified liposomes and serum proteins as evidenced by fluorescence assays. Significantly, this new form of LSNA exhibits more rapid cellular uptake and increased sequence‐specific toll‐like receptor activation in immune reporter cell lines, making it a promising candidate for immunotherapy.