(M1230-10-57) Formulation of an Oil-in-Hydrogel Anti-Adhesion System for Sustained Release of Liposomes-Encapsulated 5-Fluorouracil

Purpose: This study develops an emulsion formulation encapsulating 5-FU in liposomes with mucoadhesive polymers and soybean oil to prevent adhesion after colon cancer surgery, aiming to reduce peritoneal and organ metastases, thereby improving patient quality of life.

Methods: Preparation of 5-FU loaded liposomes: Liposomes were prepared by dissolving cholesterol, lipoid E80, and 5-FU in methylene chloride (Table 1). After evaporating the solvent at 40°C to form a thin lipid film, 50 mL of distilled water was added at 80°C and hydrated using a bath sonicator. Size reduction of the liposomes was achieved at 80°C via three cycles of high-pressure homogenizer (Nano DeBEE, Bee International, Inc., USA) at 15,000 psi. Formulation of emulsion anti-adhesion agents: The PEG phase was prepared by dispersing Xan and Pec in PEG 400 and aqueous phase was prepared by dissolving 5-FU, PX 407, TW 80, and PEG 4,000 in distilled water (DW). Soybean oil was added to the PEG phase and was mixed at 500 rpm for 5 minutes via a direct drive stirrer (Pltech Co., Ltd., Seoul, Korea). The aqueous phase was then added to this blend and stirred at 1,000 rpm for 1 hour to form an emulsion (Table 2). Composition of anti-adhesion emulsion containing liposomes: The emulsion containing liposomes were prepared as AH0, AH5, and AH10. AH0 is consisted of 100% emulsion (E1). AH5 is a mixture of 95% emulsion (E1) and 5% liposomes loaded with 5-FU (LF2). AH10 is consisted of 90% emulsion (E1) and 10% liposomes loaded with 5-FU (LF2). These formulations were stirred at 500 rpm for 10 minutes.

Results: Analysis of Particle Size: The study measured particle size of the prepared liposomes. On average, LF1 measured 123.05 nm, and LF2 measured 131.88 nm, confirming their nanoscale sizes. Determination of Encapsulation Efficiency: The aim of this experiment was to quantify the concentration of 5-FU successfully encapsulated within the liposomes. The encapsulation efficiency (EE) was 32.01% for LE1 and 46.44% for LF2. Rheology study: Both emulsion and AH formulations showed shear-thinning behavior suitable for syringe application. Emulsion E1 had a viscosity of 350.9 Pa·s at a shear rate of 1 s⁻¹. For AH formulations, viscosity decreased with higher LF content: AH0 had 350.9 Pa·s, AH5 had 244.8 Pa·s, and AH10 had 235.3 Pa·s. Drug Release Study: In vitro drug release studies of 5-FU were performed for LF1, LF2, E1, AH0, AH5, and AH10. The release of 5-FU loaded in the liposome were ranged from 27% to 29% at 48 hours. When comparing LF1 and LF2, the cholesterol-free LF1 released 28.83%, while the drug release of the cholesterol-containing formulation, LF2, showed slower rate at/by 27.65%. At 48 hours, 42.2% of drug release was observed in E1. The drug release patterns of formulations AH0, AH5, and AH10, which were mixtures of LF2 and emulsion E1, were analyzed. In AH0, which did not contain liposomes, 42.2% of the drug was released at 48 hours. AH5 and AH10, which contained liposomes, showed drug releases of 38.8% and 33.5%, respectively, after 48 hours. These results indicate that incorporating liposomes as an anti-adhesion agent can lead to sustained drug release. In Vitro Evaluation of Residence Stability: An in vitro stability study in PBS buffer showed that AH0 (without liposomes) began to erode after 17 days, AH5 after 14 days, and AH10 after 17 days, and all formulations were almost completely eroded by day 21. Throughout the experiment, all formulations exhibited swelling, doubling their original weight. Viability of Cancer Cells: To analyze the anticancer effects of 5-FU loaded formulations, the viability of HCT 116 cancer cell line was examined. Figure 1 depicts survival rates for 5-FU solution, LF1, LF2, AH0, AH5, and AH10, demonstrated reduced cell survival as 5-FU concentration increased. Both LF and AH formulations exhibited lower survival rates compared to the 5-FU solution, particularly evident in HCT116 cells. The IC50 values for AH formulations followed AH0 < AH5 < AH10, indicating that higher ratios of LF in the emulsion corresponded to greater decreases in cancer cell survival. In Vivo Evaluation of Intraperitoneal Anti-Adhesion Effect: Intra-abdominal adhesion prevention efficacy was assessed by comparing a control group, Guardix-SG, AH0, AH5, and AH10 formulations. The control group exhibited severe adhesions (severity score: 3 ± 1.41, area score: 3.33 ± 0.94), while Guardix-SG showed milder adhesions (severity and area scores of 1.67 ± 0.94). In the AH0, AH5, and AH10 groups, there were no adhesions, which demonstrated effective preventive effects and superior efficacy compared to Guardix-SG.

Conclusion: This study encapsulated 5-FU in liposomes within emulsions for sustained release. AH0 (no liposomes) released 42.2% of the drug at 48 hours, while AH5 and AH10 (with liposomes) released 38.8% and 33.5%, respectively. Higher liposome content improved anticancer efficacy, showing promise for colon cancer surgery and metastatic cancer treatment.

Acknowledgements: This work was supported by the Mid-Career Researcher Program (No. NRF-2021R1A2C2008834) and the Basic Research Infrastructure Support Program (University-Centered Labs-2018R1A6A1A03023718) through the National Research Foundation of Korea (NRF) funded by the Korean government (MSIT).

Table 1. Formulation of 5-FU loaded liposome

Table 2. Formulation of oil-based emulsions

Figure 1. Viability of HCT116 cell lines treated with 5-FU solution, LF1, LF2, AH0, AH5 and AH10. (n=3)