(M1530-06-34) Fluorinated Polyethylene Glycol Decorated Liposomes for Mitochondria-Targeted Delivery of Heat Shock Protein Inhibitors

Purpose: Current mitochondria-targeted drug delivery employs cationic moieties, such as triphenyl phosphonium (TPP), to target the negatively charged mitochondrial membrane. However, these cations can lead to several issues, including nonspecific interactions, alterations in mitochondrial membrane potential, and disruption of vital mitochondrial processes. To address these issues, we synthesized charge-neutral fluorinated polyethylene glycol (2k Da), i.e., PEG2K-F7, which demonstrated excellent mitochondrial specificity. Apoptozole (Az) is an inhibitor of heat shock protein (HSP 70) that can promote apoptosis in cancer cells. Unfortunately, the low specificity of Az may result in off-target toxicity. This work aims to prepare PEG2K-F7-decorated liposomes (Lipo-PEG2K-F7) for mitochondria-targeted delivery of Az in cancer cells, to enhance drug specificity and efficacy while reducing drug doses.

Methods: Heptafluorobutyric anhydride was reacted with cysteine ethyl ester in chloroform in the presence of triethylamine (TEA) to obtain thiol-terminated heptafluorobutyl cysteine ethyl ester (F7-CEE). Subsequently, F7-CEE was reacted with maleimide-functionalized PEG2K (PEG2K-MAL) in dimethyl formamide (DMF) in the presence of a trace amount of TEA (Figure 1A). A 7:3 molar ratio of dipalmitoyl phosphatidylcholine (DPPC) and cholesterol (Chol) was taken to prepare the liposomes for drug (Az) encapsulation. The Az, DPPC, and Chol were dissolved in methanol and dried into a thin film. The film was hydrated with phosphate-buffered saline (PBS) and probe sonicated to form the Az-loaded liposomes. Then, the liposomal suspension was used to hydrate the thin film of PEG2K-F7, which was further extruded through a 0.1µm pore size polycarbonate membrane at 37 °C using an Avanti mini extruder for the surface modification of the liposomes, resulting in the Lipo-PEG2K-F7 (Figure 1B). The particle size of Lipo-PEG2K-F7, before and after drug loading, was determined using dynamic light scattering. The 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-(lissamine rhodamine B sulfonyl) (Rh-PE) was loaded into the liposomes to study their cellular uptake and mitochondrial localization in vitro. Mitochondrial co-localization of liposomes were determined using MitoView™ Green. Cell viability was assessed using the CellTiter-Blue® assay on the HepG2 cells.

Results: The particle size of PEG2k-F7 decorated liposomes before and after drug loading was around 105 ± 3.7 nm and 120 ± 5.4 nm, respectively (Figure 1C). The Lipo-PEG2K-PE showed negligible co-localization with mitochondria, while the Lipo-PEG2K-F7 showed clear accumulation in mitochondria (Figure 2A). CellTiter-Blue^® cell viability assay indicated a significant difference between the Lipo-PEG2K-F7 Lipo-PEG2K-PE in higher Az doses, whereas lower drug doses did not show any significant difference (Figure 2B).

Conclusion: Lipo-PEG2K-F7 is a novel approach for mitochondria-targeted delivery of HSP inhibitors. The Lipo-PEG2K-F7 increased cellular uptake and mitochondrial specificity to enhance Az efficacy and decrease Az doses. The PEG2k-F7’s neutral charge would resolve the issues associated with cationic targeting ligands, such as the nonspecific biodistribution and disruption of mitochondrial structure and processes.

References: (1) Sanchez-Aranguren, L.; Anas, A. T. M.; Muhammad, U.; Parag, J.; and Marwah, M. K. Mitochondrial-targeted liposome-based drug delivery – therapeutic potential and challenges. Journal of Drug Targeting 2025, 33 (5), 575-586.
(2) Liu, Y.; Zhang, J.; Tu, Y.; Zhu, L. Potential-Independent Intracellular Drug Delivery and Mitochondrial Targeting. ACS Nano 2022, 16 (1), 1409-1420.
(3) Cho, H. J.; Gee, H. Y.; Baek, K.-H.; Ko, S.-K.; Park, J.-M.; Lee, H.; Kim, N.-D.; Lee, M. G.; Shin, I. A Small Molecule That Binds to an ATPase Domain of Hsc70 Promotes Membrane Trafficking of Mutant Cystic Fibrosis Transmembrane Conductance Regulator. J. Am. Chem. Soc. 2011, 133 (50), 20267-20276.

Acknowledgements: This work was funded by DoD PRMRP Discovery Award (#HT9425-23-1-0119) and UT Mays Cancer Center-TAM Health Joint Pilot Grant. Conflict of interest: The authors declare no competing financial interest.


Fig. 1. (A) Synthesis of the polymer, PEG2K-F7. (B) Preparation of the Lipo-PEG2K-F7 through thin film hydration, probe sonication, and extrusion. (C) Particle size of the Lipo-PEG2K-F7 with or without drug loading as measured by DLS.


Fig. 2. (A) Cellular uptake and mitochondrial colocalization of Rh-PE loaded liposomes, Lipo-PEG2K-F7 (a) and Lipo-PEG2K-PE (b). Mitochondria were stained with MitoViewTM Green. (B) CellTiter-Blue® cell viability assay in HepG2 cells. *, p=0.05