Enhanced mRNA delivery into lymphocytes enabled by lipid-varied libraries of charge-altering releasable transporters

Purpose: Efficient mRNA delivery to lymphocytes remains a major unmet need for immunotherapy and vaccinology. Current methods rely largely on physical transfection such as electroporation, which is ex vivo, costly, and inefficient; chemical vectors generally achieve low lymphocyte mRNA transfection, especially in vivo.

Hypothesis: Combinatorial variation of the lipid domain in charge-altering releasable transporters (CARTs), including binary mixtures and hybrid-lipid CARTs, will enhance mRNA delivery into lymphocytes compared with single-lipid CARTs or commercial Lipofectamine.

Aims: Synthesize a library of CARTs with varied lipid side chains. - Screen binary/ternary CART mixtures for mRNA delivery in lymphocyte cell lines. - Identify lead lipid combinations and design single hybrid-lipid CARTs incorporating the optimal lipids. - Evaluate in vitro and in vivo lymphocyte mRNA delivery, viability, and possible mechanism.

Delivery system: Platform: Charge-altering releasable transporters (CARTs), amphiphilic poly(carbonate)-b-(α-amino ester) diblock/triblock polymers. - Mechanism: Cationic α-amino ester block binds mRNA; undergoes ester-to-amide rearrangement to neutral diketopiperazine, releasing mRNA. - Lipid domains screened: dodecyl, oleyl, benzyl, ethyl, cholesterol, nonenyl, stearyl; homooligomer dodecyl control. - Payload: mRNA encoding firefly luciferase, EGFP, or Cy5-EGFP. - Lead systems: noncovalent mixture of oleyl CART 3 and nonenyl CART 7; hybrid-lipid CARTs 9–11. - Formulation: simple mixing with mRNA, no microfluidics required.

Approach: In vitro lymphocyte lines: Jurkat (T cell), EL4 (T cell), A20 (B cell); activated primary murine T cells. - In vitro non-lymphocyte lines: HeLa, DC2.4, A549. - Screen: 64 CART mixtures at 33% and 66% in 96-well format; Fluc mRNA readout. - Optimization: varied ratios of CARTs 3 and 7 (10:0 to 0:10). - In vivo: BALB/c mice, tail-vein injection of CART-mRNA complexes; spleen lymphocyte transfection analyzed by flow cytometry.

Key methods: Bioluminescence imaging for luciferase expression. - Flow cytometry for EGFP and Cy5-mRNA uptake/transfection. - MTT assay for cell viability. - Dynamic light scattering for particle size. - Qubit RNA dye assay for mRNA encapsulation. - In vivo IVIS imaging and splenocyte flow cytometry.

Key results: Lead 1:2 mixture of O₁₁:A₉ (3) and N₁₀:A₁₀ (7) enhanced Jurkat expression 6-fold over D₁₃:A₁₁; EL4 3-fold, A20 10-fold, primary T cells 9-fold. - Optimized 5:5 mixture of CARTs 3 and 7 gave >4-fold higher expression than either CART alone. - Hybrid-lipid CARTs 9 and 11 achieved 77–81% EGFP-positive Jurkat cells vs 9% for Lipofectamine 2000 and ~56% for CART 10. - mRNA encapsulation was 96–99%; particle sizes were 177–238 nm; cell viability generally >80%. - In vivo, hybrid-lipid CART 13 transfected ~1.6% CD4 T cells, ~1.5% CD8 T cells, and ~11% B cells in spleen, outperforming single-lipid CART 12 and prior LNP systems. - Mechanism appeared mainly increased mRNA uptake, not differences in size or encapsulation.

Interpretation: Combinatorial lipid variation and hybrid-lipid CART design can substantially improve mRNA delivery to lymphocytes. The lead systems enable high in vitro transfection and higher-than-previously-reported in vivo lymphocyte transfection, supporting potential applications in cancer immunotherapy, vaccines, and ex vivo/in vivo T-cell engineering.

Limitations: In vivo lymphocyte transfection remains low in absolute terms (~1.5% T cells). - Only murine models; no human primary T cells or large-animal validation. - No disease or therapeutic efficacy model; reporter genes only. - Long-term safety and repeated-dose toxicity were not evaluated. - Mechanism of enhanced uptake is partly speculative. - Hybrid CART synthesis and scalability were not assessed clinically.