Fluorinated Nanoparticles Deliver Genes Directly Into Mitochondria to Treat Inherited Blindness

Mitochondrial DNA mutations affect roughly 1 in 4,300 people and underlie a spectrum of devastating diseases — from inherited blindness to cardiomyopathy and neurodegeneration — for which no curative treatment currently exists. The double-membrane architecture of mitochondria has long frustrated gene delivery efforts, and existing approaches that rely on cationic lipophilic groups such as triphenylphosphonium (TPP) require an intact mitochondrial membrane potential (MMP) to work — a condition often compromised in the very diseases these therapies aim to treat. This study addresses that fundamental barrier by engineering a new class of fluorinated lipid nanoparticles designed to penetrate mitochondrial membranes in an MMP-independent manner.

The research team at China Pharmaceutical University synthesized four ionizable lipid variants differing in the length of their fluorinated hydrophobic tails: non-fluorinated (0F), 4-carbon fluorinated (4F), 6-carbon fluorinated (6F), and 8-carbon fluorinated (8F). These were incorporated into 16 distinct LNP formulations by varying both lipid type and molar ratio alongside the FDA-approved ionizable lipid ALC-0315, DSPC, cholesterol, and DSPE-PEG2000. Nanoparticles were prepared via microfluidic mixing and loaded with either fluorescently labeled Cy5-pDNA or a mitochondria-specific GFP plasmid (mtGFP). The mtGFP construct was carefully engineered with a cytoplasmic stop codon and no mitochondrial targeting sequence to ensure that any GFP signal detected in mitochondria could only arise from direct mitochondrial transfection — ruling out cytoplasmic expression followed by protein import.

Systematic screening revealed that both cellular and mitochondrial uptake, as well as mtGFP transfection efficiency, followed an inverted-U relationship with increasing fluorination. The 6F formulation at a specific molar ratio achieved the best mitochondrial delivery, corresponding to a fluorine mass ratio of 7.94% of total lipids. This formulation produced mitochondrial GFP transfection efficiency approximately five times higher than LNPs without any fluorinated modification. Mechanistic studies showed that fluorinated groups interact specifically with cardiolipin — a phospholipid uniquely enriched in the inner mitochondrial membrane — enabling rapid membrane traversal. Critically, when MMP was pharmacologically collapsed with FCCP (a mitochondrial uncoupler), fluorinated LNPs maintained their mitochondrial binding and uptake while non-fluorinated, MMP-dependent systems failed. This MMP-independence represents a decisive functional advantage for therapeutic use in diseased mitochondria.

To maximize selective mitochondrial delivery, the researchers conjugated a mitochondrial targeting sequence (MTS) peptide to the optimized 6F-LNP surface, producing F-M-LNPs. In cells derived from patients with Leber's hereditary optic neuropathy (LHON) — a mitochondrial disease caused by the m.11778G>A mutation in the ND4 subunit of Complex I — F-M-LNPs achieved a 3.8-fold increase in functional ND4 protein expression compared to non-fluorinated LNPs. In a male mouse model with virally induced mtDNA mutation mimicking LHON, intravitreal injection of F-M-LNPs carrying a wild-type ND4 gene led to significant alleviation of optic neuropathy symptoms, including improvements in retinal ganglion cell survival and visual function metrics.

All fluorinated lipid formulations demonstrated cell viability above 90% in Neuro-2a cells across tested concentrations up to 60 µg/mL, confirming good biocompatibility. The study is currently limited to preclinical models, and long-term safety, immunogenicity, and biodistribution in larger animals have not yet been evaluated. Nevertheless, this work provides compelling proof of concept that incorporating fluorinated modifications into lipid vectors enables safe, efficient, and MMP-independent mitochondrial gene delivery — a strategy with broad translational potential across the spectrum of mitochondrial diseases.