Improvement of cytotoxic and apoptogenic properties of crocin in cancer cell lines by its nanoliposomal form

Figures & data

Table 1.  Composition and molar ratio of different liposomal formulations containing crocin.

DOPE	DSPG	EPC	Chol.	DSPC	Formulation type
3	—	—	3	7	I
—	3	7	7	—	II
—	3	—	7	7	III

DOPE, Dioleoylphosphatidylethanolamine; DSPG, distearoylglycerophosphoglycerol; EPC, egg phosphatidylcholine; Chol., cholesterol; DSPC, distearoylphosphatidylcholine.

Table 2.  Mean size and encapsulation efficiency of different liposomal formulations containing crocin (mean ± SD, n = 3).

	Formulation I	Formulation II	Formulation III
Mean diameter (nm)	194.86 ± 25.82	149.66 ± 0.92	158.83 ± 2.88
Encapsulation efficiency (%)	4.6 ± 0.8	4.8 ± 1.3	7.2 ± 2.2

Table 3.  Doses inducing 50% cell growth inhibition (IC₅₀) of crocin and its nanoliposomes against HeLa, MCF-7, and L929 cell lines.

Cell line Agent	HeLa	MCF-7	L929
Crocin	1.603	7.35	5.7
Liposomal formulation I	0.61	1.22	0.6
Liposomal formulation II	1.2	No inhibitory effect	No inhibitory effect
Liposomal formulation III	0.64	2.07	No inhibitory effect

Cells were treated with different concentrations of crocin and its nanoliposomes for 48 h. Viability was quantitated by 3-(4,5-dimethyl thiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay.

Figure 1.  Effect of crocin on cell viability of HeLa (A), MCF-7 (B), and L929 (C) cells. Cells were treated with different concentrations of crocin for 24, 48, and 72 h. Viability was quantitated by 3-(4,5-dimethyl thiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay. Results are mean ± SD (n = 3). The asterisks are indicator of statistical differences obtained separately at different time points compared with their controls shown in figure as *P < 0.05, **P < 0.01, and ***P < 0.001.

Figure 2.  Effect of crocin liposome 1 on cell viability of HeLa (A), MCF-7 (B), and L929 (C) cells. Cells were treated with different concentrations of crocin liposome 1 for 24, 48, and 72 h. Viability was quantitated by 3-(4,5-dimethyl thiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay. Results are mean ± SD (n = 3). The asterisks are indicator of statistical differences obtained separately at different time points compared with their controls shown in figure as *P < 0.05, **P < 0.01, and ***P < 0.001.

Figure 3.  Morphological changes of crocin and liposomal crocin in MCF-7 and HeLa cells. HeLa and MCF-7 control cells (A and B), 48 h after treatment with 1 mM of crocin (C and D) and liposomal crocin (E and F). Control cells (A and B) remained untreated although receiving an equal volume of the solvent.

Table 4.  The percent of apoptotic cells in HeLa cells after 48 h of crocin and its liposomal forms.

	Control	Crocin			Liposomal formulation I		Liposomal formulation II		Liposomal formulation II
Apoptosis (%)	3.8	1 mM	2 mM	4 mM	0.5 mM	1 mM	0.5 mM	1 mM	0.5 mM	1 mM
		8.3	10.7	30.8	45.6	61.5	26.7	71.5	42.1	80.6

Figure 4.  Flow cytometry histograms of apoptosis assays by propidium iodide (PI) method in HeLa cells after 24 and 48 h. HeLa cells were treated with (1, 2, and 4 mM) of crocin and (0.5 and 1 mM) of crocin nanoliposomes for 24 and 48 h. Sub-G1 peak as an indicative of apoptotic cells was induced in crocin and nanoliposome-treated but not in control cells.