Introduction:: The surface modification of membrane-based nanoparticles, such as liposomes, polymersomes, and lipid nanoparticles, with targeting molecules is important for the design of targeted therapeutic materials. However, modification with proteins and peptides that enhance cell targeting can be costly and time-consuming, requiring lengthy conjugation and purification steps to attach the proteins to the surface of nanocarriers. These manufacturing bottlenecks limit the development of targeted nanocarriers. To develop a rapid method for the conjugation of multiple antibodies to nanoparticles for cellular targeting, we have developed a DNA-anchoring method to modify the surface of lipid-based nanoparticles. This method enables precise control over the density and stoichiometry of multiple antibodies on the surface of nanoparticles. Furthermore, these nanoparticles can be incubated with cells in vitro to assess the efficiency of different antibody combinations. Specifically, we sought to target T cells for transfection using targeted-LNPs due to their relevance in the development of cancer immunotherapies. Using the DNA-anchoring method, we identify specific antibody combinations on the surface of LNPs that significantly enhance delivery of mRNA to T cells.
Materials and Methods:: Lipid nanoparticles (LNPs) were prepared using a lipid mixture of Dlin-MC3-DMA, distearoylphosphatidylcholine, cholesterol, and 18:0 PEG2000 PE at a 50:13:35:2 molar ratio. mRNA that encoding for firefly luciferase was encapsulated. Single strand DNA (ssDNA) with a 5’ DBCO modification was conjugated to an azide-modified phospholipid lipid using copper-free click chemistry. Complimentary DNA strands were conjugated to antibodies using an adapter Protein G which naturally binds to two sites in the IgG heavy chain (AlphaThera). (Figure 1A). For the assembly of the targeting LNP, the LNPs, Lipid-ssDNA, and antibody-cDNA were incubated together at 37℃ for 1 hour. To test transfection efficiency in T cells, Jurkats were treated with LNPs modified with various antibodies targeting different T cell markers. After 24 hours, substrate was added to cells to measure luciferase expression in a plate reader (Figure 1B).
Results, Conclusions, and Discussions:: Lipid-DNA conjugation allowed for a simple, one step method for creating libraries of LNPs modified with T cell receptor antibodies. Using this method, we chose to investigate whether targeting receptors on T cells could enhance mRNA LNP delivery. Of the LNPs modified with a single antibody, the anti-CD3-modified LNPs resulted in the highest Jurkat cell luminescence indicating successful delivery and translation of the luciferase gene (Figure 1C). Anti-CD3 binds to CD3ε which is part of the T cell receptor (TCR) complex. The binding and clustering of the TCR complexes has been shown to initiate endocytosis which is likely leading to greater internalization of the anti-CD3 modified LNPs. When a second antibody such as anti-CD28, anti-CD4, or anti-CD5 was used in combination with the CD3 antibody, we saw an approximately 2-fold increase in luciferase expression when compared to anti-CD3 alone. Anti-CD3 in combination with an additional targeting molecule likely causes a synergistic effect, increasing the delivery and translation of the luciferase mRNA to T cells. DNA directed anchoring of antibodies to lipid nanoparticles provides a rapid and modular technique for studying the interactions between cells and modified particles which will enhance the development of targeted therapeutic materials.
