Engineering hybrid nanocarriers for miRNA intracellular restoration in the treatment of T-Large Granular Lymphocyte Leukemia

Large Granular Lymphocyte Leukaemia (T-LGLL) is a rare hematological malignancy characterized by clonal expansion of cytotoxic T lymphocytes. T-LGL cells frequently harbor STAT3 mutations associated with downregulation of miR-146b, leading to increased expression of Fas ligand (Fas-L) and soluble Fas-L, which strongly correlate with neutropenia, a major clinical feature of the disease. Restoring physiological miR-146b levels has therefore emerged as a promising therapeutic strategy. The aim of this project was to develop an efficient and selective nanocarrier for targeted delivery of miR-146b to leukemic T-LGL cells. Lipid nanoparticles (LNPs) were engineered and functionalized with a Fab′ fragment specific for CD57, a surface marker selectively expressed on T-LGL cells. A stepwise formulation strategy was applied to optimize miRNA loading, cellular uptake, endosomal escape, serum stability, and biocompatibility. An LNP library was generated using a synthetic arginine-rich oligocationic enhancer (OCE, 4 mol%) to improve miRNA condensation, combined with different ionizable lipids (DOPE, DODAP, D-Lin-MC3-DMA, ALC-0315, and SM-102). OCE-containing LNPs displayed sizes around 100 nm, positive zeta potentials, and loading efficiencies of approximately 90%, almost doubling those of OCE-free formulations. Functional delivery was assessed in H1299 eGFP-expressing cells using anti-eGFP siRNA. LNPs containing OCE and last-generation ionizable lipids (D-Lin-MC3-DMA, ALC-0315, and SM-102) achieved the highest silencing efficiency (IC50 0.32–0.47 nM), whereas formulations based on fusogenic lipids showed reduced activity. These results demonstrate that OCE enhances cellular uptake, while efficient cytosolic delivery relies on its synergistic interaction with pH-responsive ionizable lipids. Based on these findings, SM-102 was selected for further development. LNPs were PEGylated using different PEG-lipids. pH-sheddable PEG-lipids provided effective charge shielding under physiological conditions while enabling PEG detachment in acidic endosomes, improving cytosolic delivery and addressing the “PEG dilemma”. Optimization of N/P ratio and PEG density identified N/P 4 and 7 mol% pH-sheddable PEG as the optimal formulation, combining high loading efficiency, biocompatibility, and prolonged circulation. In vivo studies in BALB/c mice confirmed the absence of hepatic or renal toxicity and reduced liver accumulation. For active targeting, anti-CD57 IgG (clone QA17A04) was selected, cleaved into Fab′ fragments, and conjugated to DSPE-PEG-maleimide. Targeted LNPs were obtained by post-insertion of Fab′CD57-PEG, achieving stable nanoscale particles with preserved loading efficiency. In vitro studies on Jurkat cells showed negligible uptake of non-targeted PEGylated LNPs, while Fab′CD57-functionalized LNPs displayed selective cellular association comparable to non-PEGylated systems. Overall, this work demonstrates that anti-CD57 functionalization converts stealth PEGylated LNPs into actively targeted nanocarriers while preserving biocompatibility, supporting their potential for selective miRNA delivery in T-LGLL therapy.