Microemulsion-based synergistic dual-drug codelivery system for enhanced apoptosis of tumor cells

Figures & data

Table 1 Physicochemical characterization of EC-ME formulations with different feeding ratios of VP16 to coix seed oil (w/w)

VP16/coix seed oil (w/w)	Particle size (nm)	PDI	Zeta potential (mV)	DEE (%)	DLEa (%)	DLEb (%)
1/400	38.92±0.63	0.14±0.02	−15.84±2.48	92.51±4.25	44.21±0.23	0.10±0.01
2/400	36.32±0.83	0.13±0.01	−16.72±2.09	94.81±4.25	45.67±0.63	0.21±0.01
3/400	38.03±0.42	0.12±0.01	−16.05±1.96	96.27±2.22	46.62±0.98	0.31±0.01
4/400	35.19±1.16	0.12±0.01	−15.34±2.25	97.01±2.89	45.48±1.35	0.43±0.01

Notes:

a Total drug loading efficiency represents anticancer drug loading capacity of the microemulsion (both VP16 and coix seed oil were considered as encapsulated drugs);

b VP16 loading efficiency represents the VP16-loading capacity of the microemulsion. All results in this table are Mean ± SD, n=3.

Abbreviations: DEE, drug entrapment efficiency; DLE, drug loading efficiency; PDI, polydispersity index.

Figure 1 The influence on size and zeta potential after introduction of VP16 into microemulsions (n=4).

Abbreviation: MEs, microemulsions.

Figure 2 Release profile of EC-MEs at various pH environments (n=3). **P<0.01 vs pH 7.4.

Abbreviation: EC-MEs, etoposide-loaded coix seed oil microemulsions.

Figure 3 Morphologic evaluation by TEM images.

Notes: A1, B1, C1 and D1 represents the morphologic alterations of EC-MEs after treatment at pH 5.5, 4.5, 6.5 and 7.4 for 0 hour, respectively; A2, B2, C2 and D2 represents the morphologic alterations of EC-MEs after treatment at pH 5.5, 4.5, 6.5 and 7.4 for 48 hours, respectively.

Abbreviation: EC-MEs, etoposide-loaded coix seed oil microemulsions.

Figure 4 Antiproliferative effects of various VP16 formulations (A) and (B) coix seed oil against A549 cells for 24 h by MTT method (n=6). In this study, the dotted line indicates less than 60% viability, defined as cytotoxic.

Abbreviation: EC-MEs, etoposide-loaded coix seed oil microemulsions.

Figure 5 A549 cellular uptake studies.

Notes: (A) Images of FITC accumulation in A549 cells by flow cytometry for 4 h. (B) Corresponding fluorescence of various FITC formulations internalization by flow cytometry (n=3). *P<0.05 and **P<0.01 vs free FITC. (C) Fluorescent images of EC-MEs endocytosis: a, free FITC; b, FITC and coix oil; c, FITC-MEs and d, e, FITC-CMEs (5 μM and 10 μM). (D) Accumulation of VP16 in A549 cells measured by HPLC at different VP16 concentrations for 4 h (n=4). **P<0.01 vs free VP16.

Abbreviations: FITC-CMEs, FITC-loaded coix seed oil microemulsions; FITC-MEs, FITC-loaded microemulsions; EC-MEs, etoposide-loaded coix seed oil microemulsions; E-MEs, etoposide-loaded microemulsions.

Figure 6 Endocytosis pathways of E-MEs, EC-MEs and the mixture of coix seed oil and VP16 in the absence or presence of various endocytosis inhibitors at the VP16 concentration of 200 μg/mL (n=4; *P<0.05 vs control).

Abbreviations: EC-MEs, etoposide-loaded coix seed oil microemulsions; E-MEs, etoposide-loaded microemulsions.

Figure 7 A549 cells apoptosis studies. (A–C) Investigation of apoptosis induction after co-incubation of cells and various VP16 formulations at different concentrations (2 μg/mL, 10 μg/mL and 20 μg/mL) for 5 h. (D) Total apoptosis ratio of A549 cells induced by EC-MEs using the Annexin V-PE/7AAD staining assay (n=3). ^##P<0.01 vs free VP16; **P<0.01 vs E-MEs.

Abbreviation: EC-MEs, etoposide-loaded coix seed oil microemulsions.

Figure S1 Transparency and corresponding particle size of various microemulsions containing different amounts of RH40.

Notes: (A) 40%, (B) 35%, (C) 30%, (D) 15%, (E) 10%, and (F) 5%. The mixture of coix seed oil and VP16 (sample F emulsion formulation) with 5% RH40 was prepared by vigorous mechanical stirring for 30 minutes.

Figure S2 Cellular uptake kinetics.

Notes: (A) Cellular uptake of EC-ME formulations with different VP16 concentrations. (B) Cellular uptake of EC-ME formulations after treatment for different time periods. (C) Cytotoxicity of EC-ME formulations with different VP16 concentrations against A549 cells. (D) Size alteration of EC-ME formulation (200 μg/mL) after treatment at various pH values for 4 hours.

Abbreviations: E-ME, VP16-loaded conventional microemulsion; EC-ME, VP16-loaded coix seed oil microemulsion.

Figure S3 Studies of apoptosis in A549 cells. Early apoptosis of A549 cells induced by EC-ME formulation using the Annexin V-PE/7-AAD staining assay (n=3). ^##P<0.01 versus free VP16; **P<0.01 versus E-ME formulation.

Abbreviations: 7-AAD, 7-aminoactinomycin; EC-ME, VP16-loaded coix seed oil microemulsion.

Table S1 Composition of sodium hydrogen phosphate and sodium dihydrogen phosphate buffer solution at various pH values

pH	0.2 mol/L Na2HPO4 (mL)	0.3 mol/L NaH2PO4 (mL)	pH	0.2 mol/L Na2HPO4 (mL)	0.3 mol/L NaH2PO4 (mL)
5.8	8.0	92.0	7.0	61.0	39.0
5.9	10.0	90.0	7.1	67.0	33.0
6.0	12.3	87.7	7.2	72.0	28.0
6.1	15.0	85.0	7.3	77.0	23.0
6.2	18.5	81.5	7.4	81.0	19.0
6.3	22.5	77.5	7.5	84.0	16.0
6.4	26.5	73.5	7.6	87.0	13.0
6.5	31.5	68.5	7.7	89.5	10.5
6.6	37.5	62.5	7.8	91.5	8.5
6.7	43.5	56.5	7.9	93.0	7.0
6.8	49.5	51.0	8.0	94.7	5.3
6.9	55.0	45.0

Note: Bold text denotes the compostitons of PBS or HAc-NaAc buffer solution at pH 4.5, 5.5, 6.5, 7.4 that were employed in the manuscript.

Table S2 Composition of acetic acid and sodium acetate buffer solution at various pH values

pH	0.2 M NaAc (mL)	0.3 M HAc (mL)	pH	0.2 M NaAc (mL)	0.3 M HAc (mL)
2.6	0.75	9.25	4.8	5.90	4.10
3.8	1.20	8.80	5.0	7.00	3.00
4.0	1.80	8.20	5.2	7.90	2.10
4.2	2.65	7.35	5.4	8.60	1.40
4.4	3.70	6.30	5.5	9.10	0.90
4.5	4.90	5.10	5.8	9.40	0.60

Note: Bold text denotes the compostitons of PBS or HAc-NaAc buffer solution at pH 4.5, 5.5, 6.5, 7.4 that were employed in the manuscript.