Folate-mediated targeted and intracellular delivery of paclitaxel using a novel deoxycholic acid-O-carboxymethylated chitosan–folic acid micelles

Figures & data

Table 1 Properties of drug-loaded micelles (n = 3)

Polymer	Feed weight ratio (drug:polymer (w/w))	EE (%)	DL (%)	Mean particle diameter (nm)	Zeta potential (mV)
DOMC-FA	5:25	92.21 ± 1.43	15.57 ± 0.63	224.8 ± 11.2	−19.4 ± 1.1
	10:25	89.34 ± 2.51	26.33 ± 0.74	241.1 ± 9.1	−18.8 ± 1.2
	15:25	84.43 ± 3.66	33.61 ± 0.96	258.2 ± 12.4	−17.3 ± 0.7
	20:25	66.32 ± 1.74	34.67 ± 1.21	265.7 ± 17.9	−16.9 ± 0.9
DOMC	10:25	70.31 ± 1.02	21.93 ± 1.34	322.5 ± 22.6	−18.1 ± 1.3
	15:25	64.66 ± 1.06	27.95 ± 0.33	356.6 ± 40.7	−16.6 ± 0.6

Abbreviations: DL, drug-loading efficiency; DOMC, deoxycholic acid-O-carboxymethylated chitosan; EE, encapsulation efficiency; FA, folic acid.

Figure 1 TEM micrographs showing surface property of drug-loaded micelles. (A) DOMC/PTX; (B) DOMC-FA/PTX.

Abbreviations: DOMC, deoxycholic acid-O-carboxymethylated chitosan; FA, folic acid; PTX, paclitaxel; TEM, transmission electron microscopy.

Figure 2 WAXD spectra of (A) PTX; (B) DOMC + PTX; (C) DOMC-FA + PTX; (D) DOMC; (E) DOMC-FA; (F) DOMC/PTX; (G) DOMC-FA/PTX.

Abbreviations: DOMC, deoxycholic acid-O-carboxymethylated chitosan; FA, folic acid; PTX, paclitaxel; WAXD, wide-angle X-ray diffraction.

Figure 3 In vitro release of PTX from micelles in PBS (pH 7.4 and 6.2) containing 0.5% w/v Tween 80. In vitro release kinetics were carried out at 37°C ± 0.5°C by the dialysis bag technique. PTX release from stock solution was investigated as a control.

Note: Data as mean ± SD, n = 3.

Abbreviations: DOMC, deoxycholic acid-O-carboxymethylated chitosan; FA, folic acid; PBS, phosphate-buffered saline; PTX, paclitaxel; SD, standard deviation.

Figure 4 Uptake of free rhodamine B (R-B), R-B-loaded DOMC, and DOMC-FA micelles and effect of free FA on uptake of the two micelles in MCF-7 cells at different times. (A) free R-B; (B) DOMC/R-B; (C) DOMC-FA/R-B; (D) DOMC/R-B plus 1 mM free FA; (E) DOMC-FA/R-B plus1 mM free folic acid.

Abbreviations: DOMC, deoxycholic acid-O-carboxymethylated chitosan; FA, folic acid; PBS, phosphate-buffered saline; PTX, paclitaxel.

Table 2 The IC₅₀ values (nmol/L) of various formulations of PTX against MCF-7 cells for 24, 48, and 72 hours

Formulations	24 hours	48 hours	72 hours
Free PTX	23.54 ± 1.3	19.02 ± 0.8	14.01 ± 0.5
Taxol	20.52 ± 0.7*	14.38 ± 1*	10.67 ± 1.1**
DOMC/PTX	22.46 ± 1.2*	14.24 ± 0.4**	11.78 ± 0.8**
DOMC-FA/PTX	15.48 ± 0.9*,#	9.89 ± 0.6**,##	6.61 ± 0.9**,##

* Notes: P < 0.05;

** P < 0.01: significantly different from the control group (free PTX);

# P < 0.05;

## P < 0.01: significantly different from the DOMC/PTX formulation. Data as mean ± SD, n = 10 wells.

Abbreviations: DOMC, deoxycholic acid-O-carboxymethylated chitosan; FA, folic acid; PTX, paclitaxel; SD, standard deviation.

Figure 5 Cytotoxicity of different formulations containing PTX against MCF-7 cells after treatment for 72 hours. Cells treated with growth medium and blank micelles were used as respective controls.

Notes: Data as mean ± SD, n = 10 wells. *P < 0.05; **P < 0.01: significantly different from the control group (free PTX); ^#P < 0.05; ^##P < 0.01: significantly different from the DOMC/PTX formulation.

Abbreviations: DOMC, deoxycholic acid-O-carboxymethylated chitosan; FA, folic acid; PTX, paclitaxel; SD, standard deviation.

Figure 6 PTX induced morphology changes in MCF-7 cells. Cells were treated with PTX in solution or in drug-loaded micelles containing total PTX concentration of 20 nM for 48 hours. Then, PI staining assay was examined. Right panels indicate cells visualized in the fluorescence mode; left panels indicate the same fields in the bright field mode. (a) and (A) Control; (b) and (B) PTX solution; (c) and (C) Taxol; (d) and (D) DOMC/PTX; (e) and (E) DOMC-FA/PTX.

Abbreviations: DOMC, deoxycholic acid-O-carboxymethylated chitosan; FA, folic acid; PI, propidium iodide; PTX, paclitaxel.

Figure 7 Nuclear morphologies of MCF-7 cells using Hoechst 33342 staining. Cells were treated with PTX in solution or in drug-loaded micelles containing total PTX concentration of 20 nM for 48 h. (A) Control; (B) PTX solution; (C) Taxol; (D) DOMC /PTX; (E) DOMC-FA/PTX.

Abbreviations: DOMC, deoxycholic acid-O-carboxymethylated chitosan; FA, folic acid; PTX, paclitaxel.