Engineering Boronic Acid-Integrated Lipid Nanoparticles for Precision Glycan-Mediated Gene Transfer

Abstract

Targeting aberrant cell-surface glycosylation offers a powerful yet underexplored strategy for achieving cell-selective gene delivery. Here, we engineer boronic acid-integrated tocopherol lipids bearing distinct glycan-recognizing headgroups, phenylboronic acid in TNB and pyridinylboronic acid in TNBPY and incorporate them as functional dopants into a previously reported tocopherol-based cationic gemini lipid formulation (DOPE-TH8S). Doping with these boronic-acid lipids produced stable co-liposomal nanoparticles (DOPE:TH8S:TNB = 2:1:1 and DOPE:TH8S:TNBPY = 2:1:1) capable of strong pDNA complexation, as verified by zeta potential measurements, ethidium bromide intercalation-reintercalation assays, and agarose gel electrophoresis. The optimized formulations showed exceptional EGFP transfection in 4T1 and MCF-7 cancer cells at an ultralow N/P ratio of 2, significantly outperforming the commercial reagent Lipofectamine 2000. Notably, the phenylboronic acid-based TNB formulation exhibited markedly higher transfection efficiency than the pyridinylboronic acid-based TNBPY system, highlighting the critical influence of headgroup electronics and binding-release affinity on glycan-mediated internalization. Blocking experiments with pre-saturated boronic acid moieties in the lipid nanoparticles greatly reduced transfection efficiency in MCF-7 cells, confirming the sialic acid-targeted uptake pathway. Furthermore, both formulations showed negligible transfection in non-cancerous McCoy cells while retaining high activity in cancer cells, demonstrating intrinsic glycan-selective targeting. Overall, this study establishes phenyl- and pyridinylboronic acid-functionalized tocopherol lipids as a new class of programmable glycan-targeting nanocarriers. By exploiting reversible boronate-sialic acid interactions, these precision-engineered co-liposomal systems achieve potent, cancer-selective gene transfer with significant implications for next-generation targeted gene therapeutics.