(T1030-07-47) Fluorinated Cationic Polymers as Nanocarriers for Intracellular Delivery of Genes and Proteins

Purpose: The effective and safe delivery of therapeutic genes and proteins remains a significant challenge in the clinical translation and drug development. This study aims to develop and evaluate two novel cationic polymer systems, fluorinated protamine (PRM-F7) and fluorinated polyethylenimine (PEI-F7), for improving macromolecule delivery efficiency, efficacy, and biocompatibility.

Methods: PRM-F7 and PEI-F7 were synthesized using carbodiimide chemistry. The carboxyl group of heptafluorobutyric acid was activated with EDC/NHS, then reacted with protamine sulfate (in HEPES buffer) or PEI (in methanol). The reactions were carried out overnight at 4 °C. After completion, the products were purified by dialysis to remove unreacted materials (Figure 1). The protein complexation efficiency was evaluated using FITC-labeled bovine serum albumin (BSA) as a model molecule through polyacrylamide gel retardation assays. The complexation with the green fluorescent protein (GFP) plasmid was characterized by particle size using dynamic light scattering (DLS), zeta potential using phase analysis light scattering (PALS), and agarose gel retardation assays. In vitro cytotoxicity, cellular uptake, and transfection studies were performed in HepG2 cells by flow cytometry and fluorescence microscopy.

Results: PRM-F7 and PEI-F7 demonstrated their enhanced complexation with plasmids and proteins, as evidenced by the significant retardation in gel migration (Figure 2). Particle size and zeta potential analyses revealed that these complexes had favorable nanoparticle size (~150–200 nm) and surface charge for efficient cellular uptake. Both polymer systems showed low cytotoxicity (data not shown). In vitro cellular uptake and transfection results demonstrated significantly higher GFP expression (Figure 3A, B) as well as protein uptake (Figure 3C) with PRM-F7 compared to PEI-F7 and non-fluorinated polymers.

Conclusion: The fluorinated cationic polymers, particularly PRM-F7, demonstrated the excellent complexation (drug loading) ability, low cytotoxicity, high cellular uptake, and high transfection efficiency, suggesting their great potential as effective intracellular delivery nanocarriers. Further studies will focus on optimizing these systems for in vivo applications and evaluating their efficacy in preclinical disease models.

References: J, Lv., et. al., (2022). Fluorination Promotes the Cytosolic Delivery of Genes, Proteins, and Peptides. Accounts of chemical research, 55(5), 722–733. DOI: https://doi.org/10.1021/acs.accounts.1c00766.
Xin, J., et. al., (2024). Fluorinated Organic Polymers for Cancer Drug Delivery. Advanced Materials, 36, 2404645. DOI: https://doi.org/10.1002/adma.202404645

Acknowledgements: This work was funded by DoD PRMRP Discovery Award (#HT9425-23-1-0119) and TAM Health Seedling Grant. Conflict of Interest: Authors have no conflict of interests.


Figure 1: Schematic illustration of the synthesis procedure for PRM-F7 and PEI-F7.


Figure 2: Complexation of PEI-F7 and PRM-F7 with the protein (FITC-BSA) (A and C) and gene (plasmid) (B and D), evaluated by gel retardation assay. PEI-F7(1:1) and PEI-F7(1:5) represent two variants of fluorinated PEI with the PEI/F7 molar ratio of 1:1 and 1:5, respectively. OC, open circular; L, linear; and SC, supercoiled forms of plasmid DNA.


Figure 3: (A) Transfection of PEI-F7 or PRM-F7 and GFP plasmid complexes, visualized by fluorescence microscopy. (B) Quantification of GFP-positive cells by flow cytometry, indicating the transfection efficiency. (C) Intracellular delivery of FITC-labeled BSA by PEI-F7 or PRM-F7, as observed by fluorescence microscopy. Cell line: HepG2 cells.