In situ delivery of thermosensitive gel-mediated 5-fluorouracil microemulsion for the treatment of colorectal cancer

Figures & data

Table 1 Characteristics of 5FU microemulsion

Mean diameter (µm)	Viscosity (cP)	Variance (PDI)	Drug content (% w/v)	Encapsulation efficiency (%)	Zeta potential
2.12±0.33	9.11±1.03	0.21±0.011	4.07±0.12	84.23±0.62	+2.01±0.11

Note: Values are presented as mean ± SD of three independent experiments.

Abbreviations: 5FU, 5-fluorouracil; PDI, polydispersity index; SD, standard deviation.

Figure 1 5FU microemulsion image in TEM.

Note: The 5FU microemulsion particles were visualized under TEM for morphological feature and size distribution (×6,000).
Abbreviations: 5FU, 5-fluorouracil; TEM, transmission electron microscopy.

Table 2 Physicochemical properties of the drug-free thermosensitive gel and TG-5FU-ME

Samples	Gel temperature (°C)	Gel strength (g)	Coverage area (cm^2/mL)	Bioadhesive force (N)
Thermosensitive gel	54.5±2.7	11.21±2.27	1.42±0.13	1.14±0.13
TG-5FU-ME	19.2±1.2	30.14±2.54	0.81±0.11	4.18±0.37

Note: Values are presented as mean ± SD of three independent experiments.

Abbreviations: TG-5FU-ME, thermosensitive gel-mediated 5FU water-in-oil microemulsion; SD, standard deviation.

Table 3 Stability of 5FU microemulsion

Examination items	Freshly prepared	24 hours	72 hours	1 week
Diameters (µm)	2.67±0.31a	10.22±1.27	20.14±1.73	ND
In visual appearance	Homogeneous	Layering	Layering	Layering, oil precipitation
5FU content (%)	99.24±1.31	101.04±2.21	99.27±1.54	100.68±1.17

Note:

a Values are presented as mean ± SD of three independent experiments.

Abbreviations: 5FU, 5-fluorouracil; ND, not detectable; SD, standard deviation.

Table 4 Stability of TG-5FU-ME

Examination items	Freshly prepared	1 month	2 months	3 months	4 months
Diameters (µm)	2.51±0.49a	2.56±0.60	2.61±0.37	2.73±0.73	2.67±0.57
In visual appearance	Homogeneous	Homogeneous	Homogeneous	Homogeneous	Homogeneous
5FU content (%)	101.35±1.11	99.24±2.04	98.91±2.34	99.69±1.08	99.83±2.04

Note:

a Values are presented as mean ± SD of three independent experiments.

Abbreviations: 5FU, 5-fluorouracil; TG-5FU-ME, thermosensitive gel-mediated 5FU water-in-oil microemulsion; SD, standard deviation.

Figure 2 Appearance of 5FU microemulsion.

Notes: 5FU microemulsion stored at 20°C±1°C was examined right after the preparation (A, E), as well as after storage for 24 hours (B, F), 72 hours (C, G), and 1 week (D, H). (A–D) Naked eye examination; (E–H) microscopic examination.
Abbreviation: 5FU, 5-fluorouracil.

Figure 3 Appearances of TG-5FU-ME.

Notes: Freshly prepared TG-5FU-ME samples were placed at 20°C±1°C for 0 hour (A, A′, F), 1 month (B, B′, G), 2 months (C, C′, H), 3 months (D, D′, I), and 4 months (E, E′, J), followed by naked eye and microscopic examination. (A–E) Naked eye examination for the solid form of TG-5FU-ME at RT; (A′–E′) naked eye examination for the liquid form sample at temperature below 10°C; (F–J) microscopic examination. 1000× magnification.
Abbreviations: RT, room temperature; TG-5FU-ME, thermosensitive gel-mediated 5FU water-in-oil microemulsion.

Figure 4 Release profiles of 5FU formulations in vitro.

Notes: Release features of 5FU in 5FU solution, 5FU thermosensitive gel, 5FU microemulsion, and TG-5FU-ME were measured in a dynamic dialysis assay.
Abbreviations: 5FU, 5-fluorouracil; TG-5FU-ME, thermosensitive gel-mediated 5FU water-in-oil microemulsion.

Figure 5 AP to BL (A→B) and BL to AP (B→A) transport of 5FU.

Notes: Experiments were performed at 37°C for 150 minutes for both directions. The donor chambers were filled with 1.5 mL of a sample solution containing 5FU (200 µM; mean ± SD, n=3). A represents 5FU solution; B represents 5FU micro emulsion; C represents TG-5FU-ME.
Abbreviations: AP, apical; BL, basolateral; 5FU, 5-fluorouracil; SD, standard deviation.

Figure 6 Formulation change affects transportation efficacy of 5FU crossing the Caco-2 cell monolayers.

Notes: The donor chambers were filled with 1.5 mL of a sample solution containing 5FU (200 µM), and the receiver compartments were filled with 2.6 mL of blank HBSS. Samples were taken at receiver side. The determination of 5FU in transport buffer was carried out by HPLC. The apparent permeability coefficient (P_app; cm/s) for 5FU was calculated according to the equation: P_app= (1/AC₀) (dQ/dt; mean ± SD, n=3).
Abbreviations: 5FU, 5-fluorouracil; HBSS, Hank’s buffered salt solution; HPLC, high-performance liquid chromatography; TG-5FU-ME, thermosensitive gel-mediated 5FU water-in-oil microemulsion; SD, standard deviation.

Figure 7 Absorption of 5FU in vitro.

Notes: Caco-2 cells were exposed to 5FU thermosensitive gel, 5FU microemulsion, or TG-5FU-ME (containing 5FU, 200 µM). The percentage of 5FU in AP and BL media and cell monolayer was determined in 2.5 hours (mean ± SD, n=3).
Abbreviations: AP, apical; BL, basolateral; 5FU, 5-fluorouracil; TG-5FU-ME, thermosensitive gel-mediated 5FU water-in-oil microemulsion; SD, standard deviation.

Figure 8 Rectal retention time.

Notes: 5FU microemulsion and TG-5FU-ME with 0.1%Cy7NHS ester were administered into the rectum, 0.5 cm above the anus using a stomach probe needle. (A–C) 5FU microemulsion (2 minutes, 5 minutes, 10 minutes after administration); (D–F) TG-5FU-ME (2 minutes, 2 hours, 5 hours after administration).
Abbreviations: 5FU, 5-fluorouracil; TG-5FU-ME, thermosensitive gel-mediated 5FU water-in-oil microemulsion; cpx, count per second.

Figure 9 Tissue distribution of 5FU.

Notes: 5FU concentrations in rectal tissue (A), regional lymph node (B), and systemic blood (C) after rectal route administration of 20 mg kg⁻¹ TG-5FU-ME (Image), 5FU microemulsion (Image), or 5FU thermosensitive gel (Image). *P<0.05 (TG-5FU-ME vs thermosensitive gel; mean ± SD, n=3).
Abbreviations: 5FU, 5-fluorouracil; TG-5FU-ME, thermosensitive gel-mediated 5FU water-in-oil microemulsion; SD, standard deviation.

Figure 10 The morphology of rectal tissues after exposure to 5FU thermosensitive gel and TG-5FU-ME.

Notes: The rat’s rectal tissue was taken 12 hours after TG-5FU-ME (A) and 5FU thermosensitive gel (B) administration (20 mg kg⁻¹ of 5FU) through rectal route. Tissue slides were examined with H&E staining (×200).
Abbreviations: 5FU, 5-fluorouracil; H&E, hematoxylin and eosin; TG-5FU-ME, thermosensitive gel-mediated 5FU water-in-oil microemulsion.