Antitumor activity of folate-targeted, paclitaxelloaded polymeric micelles on a human esophageal EC9706 cancer cell line

Figures & data

Figure 1 Cell growth inhibition efficacy of the drug formulations on EC9706 cells after incubation for 24, 48, and 72 hours.

Notes: The equivalent PTX concentrations were 0.01, 0.1, 1, 10, and 100 μg/mL. Data are presented as the mean ± standard deviation.

Abbreviations: PTX, paclitaxel; M(PTX), paclitaxel-loaded micelles; FA-M(PTX), folate-targeted, paclitaxel-loaded micelles.

Figure 2 Apoptosis of EC9706 cells detected by flow cytometry at 24, 48, and 72 hours.

Notes: At 24 hours, ^#P < 0.01 versus controls, there was no significant difference between the treatment groups. At 48 hours, ^#P < 0.01 versus controls; *P < 0.05, compared with FA-M(PTX). At 72 hours, ^#P < 0.01, versus controls; ^▲P < 0.01, versus FA-M(PTX).

Abbreviations: PTX, paclitaxel; M(PTX), paclitaxel-loaded micelles; FA-M(PTX), folate-targeted, paclitaxel-loaded micelles.

Figure 3 (A) CLSM images and flow cytometry analysis of EC9706 cells incubated with free RhB (a), M(RhB) (b) and FA-M(RhB) (c) in folic acid-free RMPI 1640 medium, respectively; (d and e) represent EC9706 cells incubated with M(RhB) and FA-M(RhB) in RMPI 1640 medium containing 1 mM of folic acid, respectively. (B) Statistical data for cellular uptake obtained by flow cytometry.

Note: *P < 0.05 versus group A.

Abbreviations: CLSM, confocal laser scanning microscopy; PTX, paclitaxel; M(PTX), paclitaxel-loaded micelles; FA-M(PTX), folate-targeted, paclitaxel-loaded micelles; FCM, flow cytometry; RhB, rhodamine B.

Figure 4 Volume change in xenograft tumors as a function of time.

Abbreviations: PTX, paclitaxel; M(PTX), paclitaxel-loaded micelles; FA-M(PTX), folate-targeted, paclitaxel-loaded micelles.

Figure 5 Excised xenograft tumors on day 12 after initial treatment. (A) controls, (B) paclitaxel, (C) paclitaxel-loaded micelles, and (D) folate-targeted, paclitaxel-loaded micelles.

Table 1 Mean ± standard deviation for volume and net weight of EC9706 xenograft tumors on day 12 after initial treatment

Group	n	Volume (mm^3)	TIR (%)	Net weight (g)	TIR (%)
Control	14	592 ± 264▲		0.54 ± 0.22▲
Paclitaxel	14	383 ± 175*,▲	33.7	0.42 ± 0.22*,▲	21.8
M(PTX)	14	365 ± 198*,▲	40.9	0.30 ± 0.15*	44.2
FA-M(PTX)	15	151 ± 132*	74.1	0.21 ± 0.16*	61.6

Notes: Volume data:

* P < 0.01 versus control;

▲ P < 0.01 versus FA-M(PTX); P = 0.812, paclitaxel versus M(PTX); weight data,

* P < 0.01 versus control;

▲ P < 0.01 versus FA-M(PTX); P = 0.107, control versus paclitaxel; P = 0.097, paclitaxel versus M(PTX); P = 0.189, M(PTX) versus FA-M(PTX).

Abbreviations: n, number of mice; TIR, tumor inhibitory rate; M(PTX), paclitaxel-loaded micelles; FA-M(PTX), folate-targeted, paclitaxel-loaded micelles.

Figure 6 Apoptosis of the xenograft tumors detected by flow cytometry on day 12 after initial treatment. (A) controls, (B) paclitaxel, (C) paclitaxel-loaded micelles, and (D) folate-targeted, paclitaxel-loaded micelles.

Figure 7 (A) Apoptosis detected by TUNEL and expression of caspase 3, Bax, and Bcl2 detected by immunohistochemistry in xenograft tumors. (a) Controls, (b) PTX, (c) M(PTX), and (d) FA-M(PTX). (B) For TUNEL, ^#P < 0.05 versus controls; *P < 0.05 versus FA-M(PTX); P = 0.648, PTX versus M(PTX). For caspase 3, ^#P < 0.01 versus controls; *P < 0.01 versus FA-M(PTX); P = 0.269, PTX versus M(PTX). For Bax, ^#P < 0.05, versus controls; *P < 0.05 versus FA-M(PTX); P = 0.859, PTX versus M(PTX). For Bcl-2, ^#P < 0.05 versus controls.

Abbreviations: n, number of mice; PTX, paclitaxel; M(PTX), paclitaxel-loaded micelles; FA-M(PTX), folate-targeted, paclitaxel-loaded micelles.

Figure 8 Tumor slices stained with hematoxylin and eosin. The slices were prepared from the margin of xenografts, and the necrotic area in the FA-M(PTX) group is significantly increased compared with that of the other groups. Straight arrows: tissue necrosis; curved arrows: macrophages; swallowtail arrows: lymphocytes. (A) Control, (B) PTX (C) M(PTX), and (D) FA-M(PTX).

Abbreviations: PTX, paclitaxel; M(PTX), paclitaxel-loaded micelles; FA-M(PTX), folate-targeted, paclitaxel-loaded micelles.

Table 2 Median and mean survival times for nude mice

Group	n	Median survival time		Mean survival time
		Median ± SD (days)	95% CI (days)	Mean ± SD (days)	95% CI (days)
Control▲▲	25	42.0 ± 2.5	37.1–46.9	42.8 ± 1.6	36.6–45.9
PTX*,▲▲	25	44.0 ± 1.9	40.3–47.7	48.7 ± 2.3	44.1–53.3
M(PTX)**,▲	25	57.0 ± 5.8	45.6–68.4	54.2 ± 2.6	49.0–59.3
FA-M(PTX)**	26	70.0 ± 2.5	65.0–75.0	62.2 ± 3.0	56.4–68.0

Notes: Log-rank test (Mantel-Cox):

* P < 0.05 (P = 0.03);

** P < 0.001 versus control;

▲ P < 0.05 (P = 0.025),

▲▲ P < 0.001, compared with FA-M(PTX); P = 0.078, PTX compared with M(PTX).

Abbreviations: CI, confidence interval; PTX, paclitaxel; SD, standard deviation; n, number of mice; M(PTX), paclitaxel-loaded micelles; FA-M(PTX), folate-targeted, paclitaxel-loaded micelles.

Figure 9 (A) Tumor volume change as a function of time after initial treatment in survival analysis; (B) Kaplan-Meier survival curves of human esophageal cancer xenograftbearing nude mice.

Abbreviations: PTX, paclitaxel; M(PTX), paclitaxel-loaded micelles; FA-M(PTX), folate-targeted, paclitaxel-loaded micelles

Table S1 Characteristics of M(PTX) and FA-M(PTX)

	M(PTX)	FA-M(PTX)
Formula
DLS	44 nm in diameter	58 nm in diameter
TEM	40–50 nm in diameter	45–60 nm in diameter
CMC	2.5 × 10^−3 g/L	4.0 × 10^−3 g/L
Content	25 wt% of PTX	22.5 wt% of PTX
		1.4 wt% of folic acid

Abbreviations: PTX, paclitaxel; M(PTX), paclitaxel-loaded micelles; FA-M(PTX), folate-targeted, paclitaxel-loaded micelles; DLS, dynamic light scattering; TEM, transmission electron microscopy; CMC, critical micelle concentration.