Formulation and Pharmacokinetics of Thermosensitive Stealth® Liposomes Encapsulating 5-Fluorouracil
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We optimize the encapsulation and investigate the pharmacokinetics of 5-Fluorouracil (5-FU) delivered by thermosensitive stealth® liposomes (TSLs) designed to trigger drug release upon hyperthermia using focused ultrasound (FUS).
5-FU was encapsulated into liposomes made of 1,2-Dipalmitoyl-sn-glycero-3-phosphocholine/cholesterol/1,2-Distearoyl-sn-glycero-3-phosphoethanolamine-N-PEG2000 either as a free molecule or complexed with copper-polyethylenimine. Heat-triggered drug release was evaluated using either a water bath or FUS. Formulation cytotoxicity was assessed on HT-29 cell line by MTS assay. Pharmacokinetics and biodistribution of 5-FU were evaluated in HT-29-tumor bearing mice.
5-FU was easily encapsulated using the lipid hydration method (encapsulation efficacy of 13%) but poorly retained upon dilution. 5-FU complexation with copper-polyethylenimine improved 5-FU retention into liposomes and allowed to obtain an encapsulation efficacy of 37%. At 42°C, heat-triggered 5-FU release from TSLs was 63% using a water bath and 68% using FUS, within 10 min, whereas it remained below 20% for the non-thermosensitive formulation. The MTS assay revealed that formulation toxicity arose from 5-FU and not from the excipients. In addition, 5-FU complex encapsulation into TSLs induces a reduction of the IC50 from 115 down to 49 μM. Pharmacokinetics reveals a longer circulation of encapsulated 5-FU and a more important body exposure, although tumor passive targeting is not significantly higher than free 5-FU.
Complexation of 5-FU with copper-polyethylenimine appears an interesting strategy to improve 5-FU retention into TSLs in vitro and in vivo. TSLs allow heat-triggered release of the drug within 10 min at 42°C, a reasonable time for future in vivo experiments.
KEY WORDS5-Fluorouracil copper-polyethylenimine complex focused ultrasound pharmacokinetics thermosensitive stealth® liposomes
Copper (II) acetate monohydrate
Dynamic light scattering
Enhanced permeation and retention
Fœtal bovine serum
Food and drug administration
High performance liquid chromatography
Human colorectal adenocarcinoma cells
Half maximal inhibitory concentration
Low temperature sensitive liposomes
CellTiter 96® AQueous One Solution Cell Proliferation Assay
Non thermosensitive liposomes
Phosphate buffer saline
Polyethylen glycol 2 KDa
Polyethylenimine, ethylendiamine branched
Pound per square inch
Phase transition temperature
Traditional thermosensitive liposomes
Ultraviolet/visible light domains
ACKNOWLEDGMENTS AND DISCLOSURE
This research work was funded by Agence Nationale de la Recherche (ANR-2010-TecSan-007). Institut Galien Paris-Sud is a member of the Laboratory of Excellence LERMIT supported by a grant from ANR (ANR-10-LABX-33). Authors would like to thank A. Maksimenko for help with the tumor model and H. Bénech for help with PK analysis.
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